Methods for modulating protein glycosylation profiles of recombinant protein therapeutics using monosaccharides and oligosaccharides

ABSTRACT

The present invention relates to the field of protein production, and in particular to methods and compositions for modulating glycosylation of proteins expressed in host cells.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/209,821, filed on Mar. 13, 2014, which in turn claimspriority to U.S. Provisional Patent Application Ser. No. 61/785,901,filed on Mar. 14, 2013, the entire contents of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

The instant invention relates to the field of protein (e.g., antibody orDVD-Ig) production, and, in particular, to methods and compositions forcontrolling and limiting the heterogeneity of proteins expressed in hostcells. The production of proteins for biopharmaceutical applicationstypically involves the use of cell cultures that are known to produceproteins exhibiting varying levels of heterogeneity. The basis for suchheterogeneity includes, but is not limited to, the presence of distinctglycosylation profiles in the produced proteins. For example, but not byway of limitation, such heterogeneity can be observed as an increase inhigh mannose N-glycans and NGA2F-GlcNAc species as well as a decrease infucosylated species, such as NGA2F species.

The glycosylation profile of a protein (e.g., an antibody or DVD-Ig) caninfluence its biological activity through changes in half-life due toeffects on clearance, folding, stability and antibody-dependent cellularcytotoxicity (ADCC) (Shental-Behor D. et al., (2008) PNAS 105:8256-8261;Kuhlmann M. et al., (2006) Nephrol. Dial. Transplant 21:v4-v8; Zheng K.et al., (2011) mAbs 3(6):568-576). ADCC is one mechanism responsible forthe therapeutic effect of antibodies such as the anti-CD20 IgG1rituximab and the anti-Her2/neu IgG1 trastuzumab. ADCC activity isinfluenced by the amount of fucose linked to the innermost GlcNAc of theFc region, with enhanced activity seen with a reduction in fucose (MoriK. et al., (2007) Cytotechnology 55:109-114).

Heterogeneity of protein glycosylation can be assayed by releasingoligosaccharides present on the protein of interest (e.g., an antibodyor DVD-Ig) via enzymatic digestion with, for example, N-glycanase. Oncethe glycans are released, the free reducing end of each glycan can belabeled by reductive amination with a fluorescent tag. The resultinglabeled glycans are separated by normal-phase HPLC (NP-HPLC) anddetected by a fluorescence detector for quantitation. Technologicaladvances in recombinant protein production analysis have provided uniqueopportunities for identifying the extent of glycosylation exhibited by aparticular protein population, particularly in the context oflarge-scale production of recombinant proteins.

Although such advances have allowed for the robust characterization ofprotein glycosylation, there remains a need in the art for cultureconditions and production methods that allow for control over theglycosylation profile of a protein therapeutic. Modulation of proteinglycosylation is particularly advantageous in the context of cellculture processes used for commercially produced recombinantbio-therapeutics as glycosylation has the potential to impacttherapeutic utility. Control of the glycosylation profile of atherapeutic protein (e.g., an antibody or an antigen binding fragmentthereof, or a DVD-Ig) is also critical for ensuring the production ofcomparable proteins such as biosimilars. Accordingly, there is a need inthe art for compositions and methods for the targeted modulation ofprotein glycosylation. The instant invention addresses this need byproviding compositions and methods for modulating protein glycosylation.The invention further provides methods for the targeted modulation ofmannosylated and fucosylated N-glycan species linked to a protein ofinterest (e.g., antibody or DVD-Ig).

SUMMARY OF THE INVENTION

In one aspect, the present invention provides methods of producing acomposition comprising a protein with a modulated glycosylation profile.The methods include culturing a host cell expressing the protein in cellculture media supplemented with a monosaccharide and/or anoligosaccharide, thereby producing the composition comprising theprotein with a modulated glycosylation profile as compared to a control,wherein the control is a composition comprising the protein produced byculturing a host cell expressing the protein in the same cell culturemedia but which is not supplemented with a monosaccharide and/or anoligosaccharide.

In one embodiment, the methods further comprise purifying thecomposition comprising the protein with a modulated glycosylationprofile.

In another embodiment, the protein is an antibody or antigen-bindingportion thereof. In a particular embodiment, the antibody is ananti-TNFα antibody. In yet another embodiment the anti-TNFα antibody isadalimumab, or an antigen binding fragment thereof. In yet anotherembodiment, the protein is a dual variable domain immunoglobulin(DVD-Ig). In one embodiment, the protein is selected from the groupconsisting of a TVD-Ig, a half-body and a RAB.

In one embodiment of the invention, the monosaccharide is tagatose. Inanother embodiment, the oligosaccharide is sucrose.

In one embodiment, the cell culture media is supplemented with asufficient amount of the monosaccharide, e.g., tagatose, to achieve amonosaccharide concentration selected from the group consisting of about1 mM, 10 mM, 30 mM, 50 mM and 70 mM. In a particular embodiment, themonosaccharide, e.g., tagatose, concentration is 30 mM.

In one embodiment, the cell culture media is supplemented with asufficient amount of the oligosaccharide, e.g., sucrose, to achieve anoligosaccharide concentration selected from the group consisting ofabout 1 mM, 7 mM, 10 mM, 15 mM, 30 mM, 50 mM and 70 mM. In a particularembodiment, the oligosaccharide, e.g., sucrose, concentration is 30 mM.

In another embodiment, the modulated glycosylation profile of theprotein comprises modulation of a fucosylation level and/or amannosylated N-glycan oligosaccharide level in the protein.

In another embodiment, the modulation of the fucosylation levelcomprises a decrease in the fucosylation level in the protein, e.g., adecrease in the level of NGA2F, NA1F-GlcNAc, NA1F and/or NA2F in theprotein. In a further embodiment, the decrease in the level of NGA2F,NA1F-GlcNAc, NA1F and/or NA2F is a decrease of about 0.1%, 1%, 1.2%,1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65%.

In one embodiment, the modulation of the fucosylation level comprises anincrease in the fucosylation level in the protein, e.g., an increase inthe level of NGA2F-GlcNAc, NA1F-GlcNAc, NA1F and/or NA2F in the protein.In another embodiment, the increase in the level of NGA2F-GlcNAc,NA1F-GlcNAc, NA1F or NA2F is an increase of about 0.1%, 1%, 1.2%, 1.5%,2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15% or 20%.

In one embodiment, an overall decrease in the fucosylation levelcomprises an increase or a decrease in the level of NGA2F, NGA2F-GlcNAc,NA1F-GlcNAc, NA1F and/or NA2F in the protein. In another embodiment, thedecrease in the fucosylation level is a decrease of about 1%, 1.2%,1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45% or 50%.

In another embodiment, the modulation of the mannosylated N-glycan levelcomprises an increase in the mannosylation level of the protein. In oneembodiment, the mannosylation level is an increase of about 0.1%, 1%,1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%. In yet another embodiment, theincrease in the mannosylation level comprises an increase in the levelof a high mannose N-glycan oligosaccharide selected from the groupconsisting of Man 5 glycan, Man 6 glycan, Man 7 glycan and Man 8 glycan.In one embodiment, the levels of Man 5 glycan, Man 6 glycan, Man 7glycan and/or Man 8 glycan are increased by about 0.1%, 1%, 1.2%, 1.5%,2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40, 45% or 50%.

In one embodiment of the present invention the host cell is a CHO cell.

In another aspect, the present invention provides methods of producingcompositions comprising an antibody, or antigen binding fragmentthereof, with a modulated glycosylation profile. The methods includeculturing a host cell expressing the antibody, or antigen bindingfragment thereof, in cell culture media supplemented with sucrose and/ortagatose, thereby producing the composition comprising the antibody, orantigen binding fragment thereof, with an increased level ofmannosylated N-glycans and a decreased level of fucosylated N-glycans ascompared to a control, wherein the control is a composition comprisingan antibody, or antigen binding fragment thereof, produced by culturinga host cell expressing the antibody, or antigen binding fragmentthereof, in cell culture media which is not supplemented with tagatoseand/or glucose. In one embodiment, the antibody is adalimumab, or anantigen binding fragment thereof.

In another aspect, the present invention provides methods of producingcompositions comprising an antibody, or antigen binding fragmentthereof, with a modulated glycosylation profile. The methods includeculturing a host cell expressing the antibody, or antigen bindingfragment thereof, in cell culture media supplemented with sucrose and/ortagatose, thereby producing the composition comprising the antibody, orantigen binding fragment thereof, with a 1-50% increase in the level ofmannosylated N-glycans and a 1-50% decrease in the level of fucosylatedN-glycans as compared to a control, wherein the control is a compositioncomprising an antibody, or antigen binding fragment thereof, produced byculturing a host cell expressing the antibody, or antigen bindingfragment thereof, in cell culture media which is not supplemented withtagatose and/or glucose. In one embodiment, the antibody is adalimumab,or an antigen binding fragment thereof.

In a further aspect, the present invention provides compositionscomprising a cell culture media comprising a monosaccharide and/or anoligosaccharide. In one embodiment, the monosaccharide is tagatose. Inanother embodiment, the oligosaccharide is sucrose.

In yet another aspect, the present invention provides pharmaceuticalcompositions comprising the protein compositions produced by the methodsof the invention and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides compositionscomprising a therapeutic protein with a modulated glycosylation profileproduced by the methods the invention. In one embodiment, thetherapeutic protein is an antibody.

In another aspect, the present invention provides compositionscomprising a therapeutic protein, wherein the protein comprises a 1-50%increase in the level of mannosylated N-glycans and a 1-50% decrease inthe level of fucosylated N-glycans, as compared to the control, whereinthe control is a composition comprising a protein produced by culturinga host cell expressing the protein in cell culture media which is notsupplemented with a monosaccharide and/or an oligosaccharide, In oneembodiment, the therapeutic protein is selected from the groupconsisting of an antibody, an antigen-binding portion thereof, DVD-Ig,TVD-Ig, RAB and half-body. In one embodiment, the therapeutic protein isan antibody.

In another aspect, the present invention provides methods of producingcompositions comprising an antibody, or antigen binding fragmentthereof, with a modulated glycosylation profile by culturing a host cellexpressing the antibody, or antigen binding fragment thereof, in cellculture media supplemented with sucrose and/or tagatose, therebyproducing the composition comprising the antibody, or antigen bindingfragment thereof, with a 1-30% increase in antibody-dependent cellularcytotoxicity (ADCC) response as compared to a control, wherein thecontrol is a composition comprising an antibody, or antigen bindingfragment thereof, produced by culturing a host cell expressing theantibody, or antigen binding fragment thereof, in cell culture mediawhich is not supplemented with tagatose and/or glucose. In oneembodiment the antibody is adalimumab, or an antigen binding fragmentthereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the chemical structure of sucrose, tagatose and fructose.

FIG. 2 depicts a simplified linear reaction view of the N-glycanbiosynthetic pathway in mammalian cells.

FIGS. 3A-3C depict the cell culture performance of Cell Line 1 in mediasupplemented with 30 mM, 50 mM or 70 mM sucrose. FIG. 3A: Viable celldensity. FIG. 3B: Percent viability. FIG. 3C: Harvest titer ratio.Control is unsupplemented media.

FIG. 4 depicts the N-glycan oligosaccharide results as an absolutepercent change in oligosaccharide profile of Cell Line 1 in mediasupplemented with 30 mM, 50 mM or 70 mM sucrose.

FIGS. 5A-5C depict the cell culture performance of Cell Line 1 in mediasupplemented with 30 mM, 50 mM or 70 mM tagatose. FIG. 5A: Viable celldensity. FIG. 5B: Percent viability. FIG. 5C: Harvest titer ratio.Control is unsupplemented media.

FIG. 6 depicts the N-glycan oligosaccharide results as an absolutepercent change in oligosaccharide profile of Cell Line 1 in mediasupplemented with 30 mM, 50 mM or 70 mM tagatose.

FIGS. 7A-7F depict the cell culture performance of Cell Line 1 inlaboratory-scale bioreactors with media supplemented with 50 mM sucrose.FIG. 7A: Viable cell density. FIG. 7B: pCO₂. FIG. 7C: Osmolality. FIG.7D: Percent viability. FIG. 7E: Lactate. FIG. 7F: Harvest titer ratio.Control is unsupplemented media.

FIG. 8 depicts the N-glycan oligosaccharide results as an absolutepercent change in oligosaccharide profile of Cell Line 1 inlaboratory-scale bioreactors with media supplemented with 50 mM sucrose.

FIGS. 9A-9F depict the cell culture performance of Cell Line 1 inlaboratory-scale bioreactors with media supplemented with 50 mMtagatose. FIG. 9A: Viable cell density.

FIG. 9B: pCO₂. FIG. 9C: Osmolality. FIG. 9D: Percent viability. FIG. 9E:Lactate.

FIG. 9F: Harvest titer ratio. Control is unsupplemented media.

FIG. 10 depicts the N-glycan oligosaccharide results as an absolutepercent change in oligosaccharide profile of Cell Line 1 inlaboratory-scale bioreactors with media supplemented with 50 mMtagatose.

FIGS. 11A-11C depict the cell culture performance of Cell Line 2 inmedia supplemented with 7 mM, 15 mM or 30 mM sucrose. FIG. 11A: Viablecell density. FIG. 11B: Percent viability. FIG. 11C: Harvest titerratio. Control is unsupplemented media.

FIG. 12 depicts the N-glycan oligosaccharide results as an absolutepercent change in oligosaccharide profile of Cell Line 2 in mediasupplemented with 7 mM, 15 mM or 30 mM sucrose.

FIGS. 13A-13D depict the cell culture performance of Cell Line 1 inmedia supplemented with 1 mM, 10 mM, 30 mM, 50 mM or 70 mM sucrose. FIG.13A: Viable cell density. FIG. 13B: Percent viability. FIG. 13C:Relative harvest titer compared to unsupplemented control. FIG. 13D:Absolute % change in protein oligosaccharide profile compared tounsupplemented control. (*p<0.05 on marked day or process conditionindicating a statistically significant difference compared to theunsupplemented control).

FIGS. 14A-14D depict the cell culture performance of Cell Line 1 inmedia supplemented with 1 mM, 10 mM, 30 mM, 50 mM or 70 mM tagatose.FIG. 14A: Viable cell density. FIG. 14B: Percent viability. FIG. 14C:Relative harvest titer compared to unsupplemented control. FIG. 14D:Absolute % change in protein oligosaccharide profile compared tounsupplemented control. (*p<0.05 on marked day or process conditionindicating a statistically significant difference compared to theunsupplemented control).

FIGS. 15A-15D depict the cell culture performance of Cell Line 2 inmedia supplemented with 1 mM, 30 mM, or 50 mM sucrose. FIG. 15A: Viablecell density. FIG. 15B: Percent viability. FIG. 15C: Relative harvesttiter compared to unsupplemented control. FIG. 15D: Absolute % change inprotein oligosaccharide profile compared to unsupplemented control.(*p<0.05 on marked day or process condition indicating a statisticallysignificant difference compared to the unsupplemented control).

FIGS. 16A-16D depict the cell culture performance of Cell Line 2 inmedia supplemented with 1 mM, 30 mM, or 50 mM tagatose. FIG. 16A: Viablecell density. FIG. 16B: Percent viability. FIG. 16C: Relative harvesttiter compared to unsupplemented control. FIG. 16D: Absolute % change inprotein oligosaccharide profile compared to unsupplemented control.(*p<0.05 on marked day or process condition indicating a statisticallysignificant difference compared to the unsupplemented control).

FIGS. 17A-17F depict the cell culture performance of Cell Line 1 inlaboratory-scale bioreactors with media supplemented with 50 mM sucrose,tagatose, or fructose. FIG. 17A: Viable cell density. FIG. 17B: Percentviability. FIG. 17C: Glucose concentration. FIG. 17D: Lactateconcentration. FIG. 17E: Osmolality. FIG. 17F: Relative harvest titercompared to unsupplemented control.

FIG. 18 depicts the absolute percent change in protein oligosaccharideprofile of Cell Line 1 in laboratory-scale bioreactors with mediasupplemented with 50 mM sucrose, tagatose, or fructose.

FIG. 19 is a schematic representation of various protein therapeutics(e.g., antibody, DVD-Ig, TVD-Ig, RAB, Half-body) whose glycosylationprofiles may be modulated using the methods of the present invention.

FIG. 20 depicts the absolute percent change in protein oligosaccharideprofile of Cell Line 3 in shake flask cultures with media supplementedwith 7 mM, 15 mM or 50 mM sucrose.

FIG. 21 depicts the Cr-51 release assay results for the measurement ofantibody dependent cell cytotoxicity (ADCC) for antibodies purified fromhost cell cultures supplemented with 7 mM, 15 mM or 50 mM sucrose. Thecontrol was an unsupplemented culture.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for modulatingthe glycosylation profile of a protein such as a therapeutic protein(e.g., antibody, DVD-Ig, TVD-Ig, Half-body or RAB compositions).

The present invention is based on the identification and optimization ofupstream process technologies, e.g., recombinant cell cultureconditions, for protein production, e.g., production of antibodies orantigen-binding portions thereof or DVD-Igs, resulting in the productionof protein compositions with modulated glycosylation profiles (e.g.,decreased fucosylation and/or increased mannosylation).

I. Definitions

In order that the present invention may be more readily understood,certain term are first defined.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear, however, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms, for example, those characterized by “a” or“an”, shall include pluralities, e.g., one or more impurities. In thisapplication, the use of “or” means “and/or”, unless stated otherwise.Furthermore, the use of the term “including,” as well as other forms ofthe term, such as “includes” and “included”, is not limiting. Also,terms such as “element” or “component” encompass both elements andcomponents comprising one unit and elements and components that comprisemore than one unit unless specifically stated otherwise.

Most naturally occurring peptides (or proteins) comprise carbohydrate orsaccharide moieties attached to the peptide via specific linkages to aselect number of amino acids along the length of the primary peptidechain. Thus, many naturally occurring peptides are termed“glycopeptides” or “glycoproteins” or are referred to as “glycosylated”proteins or peptides.

The term “glycoform” refers an isoform of a protein, e.g., an antibody,that differs only with respect to the number and/or type of attachedglycan(s). Glycoproteins often consist of a number of differentglycoforms.

The predominant sugars found on glycoproteins are glucose, galactose,mannose, fucose, N-acetylgalactosamine (“GalNAc”), N-acetylglucosamine(“GlcNAc”) and sialic acid (e.g., N-acetylneuraminic acid (“NANA” or“NeuAc”, where “Neu” is neuraminic acid) and “Ac” refers to “acetyl”).The processing of the sugar groups occurs co-translationally in thelumen of the ER and continues in the Golgi apparatus for N-linkedglycoproteins.

The oligosaccharide structure attached to the peptide chain is known asa “glycan” molecule. The glycan structures found in naturally occurringglycopeptides are typically divided into two classes, “N-linked glycans”or N-linked oligosaccharides” and “O-linked glycans” or 0-linkedoligosaccharides”.

Peptides expressed in eukaryotic cells typically comprise N-glycans.“N-glycans” are N-glycosylated at an amide nitrogen of an asparagine oran arginine residue in a protein via an N-acetylglucosamine residue.These “N-linked glycosylation sites” occur in the peptide primarystructure containing, for example, the amino acid sequenceasparagine-X-serine/threonine, where X is any amino acid residue exceptproline and aspartic acid.

Techniques for the determination of glycan primary structure are wellknown in the art and are described in detail, for example, in Montreuil,“Structure and Biosynthesis of Glycopeptides” In Polysaccharides inMedicinal Applications, pp. 273-327, 1996, Eds. Severian Damitriu,Marcel Dekker, NY. It is therefore a routine matter for one of ordinaryskill in the art to isolate a population of peptides produced by a celland determine the structure(s) of the glycans attached thereto. Forexample, efficient methods are available for (i) the splitting ofglycosidic bonds either by chemical cleavage such as hydrolysis,acetolysis, hydrazinolysis, or by nitrous deamination; (ii) completemethylation followed by hydrolysis or methanolysis and by gas-liquidchromatography and mass spectroscopy of the partially methylatedmonosaccharides; and (iii) the definition of anomeric linkages betweenmonosaccharides using exoglycosidases, which also provide insight intothe primary glycan structure by sequential degradation. Flouresecentlabeling and subsequent high performance liquid chromatography (HPLC),e.g., normal phase HPLC (NP-HPLC), mass spectroscopy and nuclearmagnetic resonance (NMR) spectrometry, e.g., high field NMR, may also beused to determine glycan primary structure.

Kits and equipment for carbohydrate analysis are also commerciallyavailable. Fluorophore Assisted Carbohydrate Electrophoresis (FACE) isavailable from Glyko, Inc. (Novato, Calif.). In FACE analysis,glycoconjugates are released from the peptide with either Endo H orN-glycanase (PNGase F) for N-linked glycans, or hydrazine for Ser/Thrlinked glycans. The glycan is then labeled at the reducing end with afluorophore in a non-structure discriminating manner. The fluorophorelabeled glycans are then separated in polyacrylamide gels based on thecharge/mass ratio of the saccharide as well as the hydrodynamic volume.Images are taken of the gel under UV light and the composition of theglycans is determined by the migration distance as compared with thestandards. Oligosaccharides can be sequenced in this manner by analyzingmigration shifts due to the sequential removal of saccharides byexoglycosidase digestion.

All N-linked oligosaccharides have a common “pentasaccharide core” ofMan₃GlcNAc₂. (“Man” refers to mannose; “Glc” refers to glucose; “NAc”refers to N-acetyl; and “GlcNAc” refers to N-acetylglucosamine). Thepentasaccharide core is also referred to as the “trimannose core” or the“paucimannose core”.

N-glycans differ with respect to the presence of, and/or in the numberof branches (also called “antennae”) comprising peripheral sugars suchas N-acetylglucosamine, galactose, N-acetylgalactosamine,N-acetylneuraminic acid, fucose and sialic acid that are added to theMan₃GlcNAc₂ core structure. Optionally, this structure may also containa core fucose molecule and/or a xylose molecule. For a review ofstandard glycobiology nomenclature see, Essentials of Glycobiology Varkiet al. eds., 1999, CSHL Press, the contents of which are incorporatedherein by reference.

N-glycans are classified according to their branched constituents (e.g.,oligomannose-type, complex, or hybrid). An “oligomannose-type” or “highmannose-type” N-glycan has five or more mannose residues.

A “complex-type” N-glycan typically has at least one GlcNAc attached tothe 1,3 mannose arm and at least one GlcNAc attached to the 1,6 mannosearm of a pentasaccharide core. Complex-type N-glycans may also havegalactose (“Gal”) or N-acetylgalactosamine residues that are optionallymodified with sialic acid or derivatives, e.g., N-acetyl neuraminicacid. Complex-type N-glycans may also have intrachain substitutionscomprising “bisecting” GlcNAc, and core fucose (“Fuc”). ComplexN-glycans may also have multiple antennae on the pentasaccharide coreand are, therefore, also referred to as “multiple antennary-typeglycans.”

A “hybrid-type” N-glycan comprises at least one GlcNAc on the terminalof the 1,3 mannose arm of the pentasaccharide core and zero or moremannoses on the 1,6 mannose arm of the trimannose core.

The oligomannose-type structures that may be present within thecompositions of the invention and/or may be used in the methods of theinvention are referred to herein as “M5” or “Man 5 glycan”; “M6” or “Man6 glycan”; “M7” or “Man 7 glycan”; “M8” or “Man 8 glycan”; and “M9” or“Man 9 glycan.”

In one embodiment, an M5 oligomannose-type structure has the structure(I):

In one embodiment, an M6 oligomannose-type structure has the structure(II):

In one embodiment, an M7 oligomannose-type structure has the structure(III):

In another embodiment, an M7 oligomannose-type structure has thestructure (IV):

In another embodiment, an M7 oligomannose-type structure has thestructure (V):

In one embodiment, an M8 oligomannose-type structure has the structure(VI):

In another embodiment, an M8 oligomannose-type structure has thestructure (VII):

In another embodiment, an M8 oligomannose-type structure has thestructure (VIII):

In one embodiment, an M9 oligomannose-type structure has the structure(IX):

In one embodiment, the oligomannose-type structures that may be presentwithin the compositions of the invention and/or may be used in themethods of the invention are independently selected from the groupconsisting of Man 5 glycan, Man 6 glycan, Man 7 glycan, Man 8 glycan,and/or Man 9 glycan.

In one embodiment, a multiple antennary-type structure that may bepresent within the compositions of the invention and/or may be used inthe methods of the invention is a “bianntennary oligosaccharide-typestructure”. A “bianntennary oligosaccharide-type structure” is anN-linked glycan having two branches or arms, and a core fucose withzero, one or two galactose additions on the arms. In one embodiment, a“bianntennary oligosaccharide-type structure” that may be present withinthe compositions of the invention and/or may be used in the methods ofthe invention is bisected. In one embodiment, a “bianntennaryoligosaccharide-type structure” that may be present within thecompositions of the invention and/or may be used in the methods of theinvention is a “fucosylated bianntennary oligosaccharide-typestructure”, e.g., comprises a core-substituted with fucose.

In one embodiment, a “fucosylated bianntennary oligosaccharide-typestructure” that may be present within the compositions of the inventionand/or may be used in the methods of the invention is an “asialo,fucosylated bianntennary oligosaccharide-type structure”, also referredto as an “asialo, bigalactosylated biantennary, core-substituted withfucose”, referred to herein as “NA2F.”

In another embodiment, a “fucosylated bianntennary oligosaccharide-typestructure” that may be present within the compositions of the inventionand/or may be used in the methods of the invention is a asialo,agalacto, fucosylated bianntennary oligosaccharide-type structure, alsoreferred to as an asialo, agalacto-, biantennary, core-substituted withfucose, referred to herein as “NGA2F.”

In another embodiment, a “fucosylated bianntennary oligosaccharide-typestructure” that may be present within the compositions of the inventionand/or may be used in the methods of the invention is a asialo,fucosylated bianntennary oligosaccharide-type structure, also referredto as asialo, monogalactosylated biantennary, core-substituted withfucose, referred to herein as “NA1F.”

In another embodiment, a “fucosylated bianntennary oligosaccharide-typestructure” that may be present within the compositions of the inventionand/or may be used in the methods of the invention is a asialo,agalacto, fucosylated biantennary, minus a bisecting N-acetylglucosamineoligosaccharide-type structure, also referred to as asialo, agalacto-,biantennary, core-substituted with fucose minus a bisectingN-acetylglucosamine, referred to herein as “NGA2F-GlcNAc.”

In yet another embodiment, a “fucosylated bianntennaryoligosaccharide-type structure” that may be present within thecompositions of the invention and/or may be used in the methods of theinvention is a asialo, monogalacto, fucosylated biantennary, minus abisecting N-acetylglucosamine oligosaccharide-type structure, alsoreferred to as asialo, monogalactosylated biantennary, core-substitutedwith fucose minus a bisecting N-acetylglucosamine, referred to herein as“NA1F-GlcNAc.”

In one embodiment, an NA2F fucosylated biantennary oligosaccharide-typestructure has the structure (X):

In one embodiment, an NGA2F fucosylated biantennary oligosaccharide-typestructure has the structure (XI):

In one embodiment, an NA1F fucosylated biantennary oligosaccharide-typestructure has the structure (XII):

In another embodiment, an NA1F fucosylated biantennaryoligosaccharide-type structure has the structure (XIII):

In one embodiment, an NGA2F-GlcNAc, and NA1F-GlcNAc fucosylatedbiantennary oligosaccharide-type structure has the structure (XIV):

In one embodiment, an NA1F-GlcNAc fucosylated biantennaryoligosaccharide-type structure has the structure (XV):

In one embodiment, the fucosylated biantennary oligosaccharide-typestructure is independently selected from the group consisting of NGA2F,NA1F, NA2F, NGA2F-GlcNAc, and NA1F-GlcNAc.

As used herein, a “modulated glycosylation profile” includes a profileof a composition comprising a protein (e.g., an antibody or DVD-Ig)which is modulated as compared to the glycosylation profile of acomposition comprising that same protein produced by culturing a hostcell expressing that protein in cell culture media which is notsupplemented with a monosaccharide (e.g., tagatose) and/or anoligosaccharide (e.g., sucrose). The modulated glycosylation profile mayinclude an overall increase in the level of mannosylated N-glycans andan overall decrease in the level of fucosylated N-glycans in theprotein. For example, the overall level of mannosylated N-glycans in theprotein may be increased by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%,3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. Ranges withinone or more of the preceding values, e.g., 1-10%, 1-15%, 1-20%, 1-25%,1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%,1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%,2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%,3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%,3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%, 4-20%,4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%,4-47%, 4-48%, 4-49%, 4-50% or 1-99% are contemplated by the invention.

In another example, the overall level of mannosylated N-glycanscomprises an increase in the amount or level of a high mannose N-glycanoligosaccharide. A high-mannose N-glycan has more than one mannoselinked to the non-reducing terminal of the core structure. For example,the high mannose N-glycan oligosaccharide is selected from the groupconsisting of Man 5 glycan, Man 6 glycan, Man 7 glycan and Man 8 glycan.In one embodiment, the amount or level of at least one of Man 5 glycan,Man 6 glycan, Man 7 glycan and/or Man 8 glycan is increased by about0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or 99%. Ranges within one or more of thepreceding values, e.g., 0.1-5%, 0.1-10%, 0.1-15%, 0.1-20%, 0.1-21%,0.1-22%, 0.1-23%, 0.1-24%, 0.1-25%, 0.1-26%, 0.1-27%, 0.1-28%, 0.1-29%,0.1-30%, 0.1-35%, 0.1-40%, 0.1-41%, 0.1-42%, 0.1-43%, 0.1-44%, 0.1-45%,0.1-46%, 0.1-47%, 0.1-48%, 0.1-49%, 0.1-50%, 1-5%, 1-10%, 1-15%, 1-20%,1-21%, 1-22%, 1-23%, 1-24%, 1-25%, 1-26%, 1-27%, 1-28%, 1-29%, 1-30%,1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%,1-49%, 1-50%, 2-5%, 2-10%, 2-15%, 2-20%, 2-21%, 2-22%, 2-23%, 2-24%,2-25%, 2-26%, 2-27%, 2-28%, 2-29%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%,2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%, 3-5%, 3-10%,3-15%, 3-20%, 3-21%, 3-22%, 3-24%, 3-25%, 3-26%, 3-27%, 3-28%, 2-29%,3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%,3-48%, 3-49%, 3-50%, 4-5%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%, 4-35%,4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%, 4-49%,4-50% or 0.1-99% are contemplated by the invention.

In another example, an overall decrease in the level of fucosylatedN-glycans resulting from modulation of any one of the fucosylated glycanspecies such as NGA2F-GlcNAc, NGA2F, NA1F-GlcNAc, NA1F and/or NA2F isdecreased by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%,4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. Ranges within one or more ofthe preceding values, e.g., 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%,1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%,1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%,2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%, 3-10%, 3-15%,3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%,3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%,4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%,4-49%, 4-50%, 5-10%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-41%,5-42%, 5-43, 5-44%, 5-45%, 5-46%, 5-47%, 5-48%, 5-49%, 5-50% or 1-99%are contemplated by the invention.

The term “level” with respect to protein such as an antibody, orantigen-binding fragment thereof, which is glycosylated at an N-linkedglycosylation site on the Fc region in a composition refers to therelation of one glycoform in the composition to the whole of theglycoform levels in the composition and is expressed as a percentage ofthe whole, e.g., 0-100%. The level in a composition may be an absoluteamount as measured in molecules, moles, or weight percent.

Compositions comprising varying levels of glycoforms of a protein suchas a human antibody, or antigen-binding fragment thereof, are useful inthat by varying the glycoform compositions a desired characteristics,e.g., rate of serum clearance or ADCC activity, may be achieved.

The methods of the invention can be used to produce compositions of anyprotein, such as a therapeutic protein, e.g., an antibody, anantigen-binding portion thereof, a DVD-Ig, a TVD-Ig, a RAB or ahalf-body.

The term “antibody” includes an immunoglobulin molecule comprised offour polypeptide chains, two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds. Each heavy chain is comprised of aheavy chain variable region (abbreviated herein as HCVR or VH) and aheavy chain constant region (CH). The heavy chain constant region iscomprised of three domains, CH1, CH2 and CH3. Each light chain iscomprised of a light chain variable region (abbreviated herein as LCVRor VL) and a light chain constant region. The light chain constantregion is comprised of one domain, CL. The VH and VL regions can befurther subdivided into regions of hypervariability, termedcomplementarity determining regions (CDRs), interspersed with regionsthat are more conserved, termed framework regions (FR). Each VH and VLis composed of three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The term “antigen-binding portion” of an antibody (or “antibodyportion”) includes fragments of an antibody that retain the ability tospecifically bind to an antigen (e.g., in the case of Adalimumab,hTNFα). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment comprising the VL, VH, CL and CHI domains; (ii) aF(ab′)2 fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) a Fd fragmentcomprising the VH and CHI domains; (iv) a Fv fragment comprising the VLand VH domains of a single arm of an antibody, (v) a dAb fragment (Wardet al., (1989) Nature 341:544-546, the entire teaching of which isincorporated herein by reference), which comprises a VH domain; and (vi)an isolated complementarity determining region (CDR). Furthermore,although the two domains of the Fv fragment, VL and VH, are coded for byseparate genes, they can be joined, using recombinant methods, by asynthetic linker that enables them to be made as a single protein chainin which the VL and VB regions pair to form monovalent molecules (knownas single chain Fv (scFv); see, e.g., Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883, the entire teachings of which are incorporated herein byreference). Such single chain antibodies are also intended to beencompassed within the term “antigen-binding portion” of an antibody.Other forms of single chain antibodies, such as diabodies are alsoencompassed. Diabodies are bivalent, bispecific antibodies in which VHand VL domains are expressed on a single polypeptide chain, but using alinker that is too short to allow for pairing between the two domains onthe same chain, thereby forcing the domains to pair with complementarydomains of another chain and creating two antigen binding sites (see,e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123, theentire teachings of which are incorporated herein by reference). Stillfurther, an antibody or antigen-binding portion thereof may be part of alarger immunoadhesion molecule, formed by covalent or non-covalentassociation of the antibody or antibody portion with one or more otherproteins or peptides. Examples of such immunoadhesion molecules includeuse of the streptavidin core region to make a tetrameric scFv molecule(Kipriyanov, S. M., et al. (1995) Human Antibodies and Hybridomas6:93-101, the entire teaching of which is incorporated herein byreference) and use of a cysteine residue, a marker peptide and aC-terminal polyhistidine tag to make bivalent and biotinylated scFvmolecules (Kipriyanov, S. M., et al. (1994) Mol Immunol. 31:1047-1058,the entire teaching of which is incorporated herein by reference).Antibody portions, such as Fab and F(ab′)2 fragments, can be preparedfrom whole antibodies using conventional techniques, such as papain orpepsin digestion, respectively, of whole antibodies. Moreover,antibodies, antibody portions and immunoadhesion molecules can beobtained using standard recombinant DNA techniques, as described herein.In one aspect, the antigen binding fragments are complete domains orpairs of complete domains.

The term “human antibody” includes antibodies having variable andconstant regions corresponding to human germline immunoglobulinsequences as described by Kabat et al. (See Kabat, et al. (1991)Sequences of proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242).The human antibodies of the invention may include amino acid residuesnot encoded by human germline immunoglobulin sequences (e.g., mutationsintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), e.g., in the CDRs and in particular CDR3. Themutations can be introduced using the “selective mutagenesis approach.”The human antibody can have at least one position replaced with an aminoacid residue, e.g., an activity enhancing amino acid residue which isnot encoded by the human germline immunoglobulin sequence. The humanantibody can have up to twenty positions replaced with amino acidresidues which are not part of the human germline immunoglobulinsequence. In other embodiments, up to ten, up to five, up to three or upto two positions are replaced. In one embodiment, these replacements arewithin the CDR regions. However, the term “human antibody”, as usedherein, is not intended to include antibodies in which CDR sequencesderived from the germline of another mammalian species, such as a mouse,have been grafted onto human framework sequences.

The phrase “recombinant human antibody” includes human antibodies thatare prepared, expressed, created or isolated by recombinant means, suchas antibodies expressed using a recombinant expression vectortransfected into a host cell, antibodies isolated from a recombinant,combinatorial human antibody library, antibodies isolated from an animal(e.g., a mouse) that is transgenic for human immunoglobulin genes (see,e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295, theentire teaching of which is incorporated herein by reference) orantibodies prepared, expressed, created or isolated by any other meansthat involves splicing of human immunoglobulin gene sequences to otherDNA sequences. Such recombinant human antibodies have variable andconstant regions derived from human germline immunoglobulin sequences(see, Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242). In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.In certain embodiments, however, such recombinant antibodies are theresult of selective mutagenesis approach or back-mutation or both.

An “isolated antibody” includes an antibody that is substantially freeof other antibodies having different antigenic specificities (e.g., anisolated antibody that specifically binds hTNFα is substantially free ofantibodies that specifically bind antigens other than hTNFα). Anisolated antibody that specifically binds hTNFα may bind TNFα moleculesfrom other species. Moreover, an isolated antibody may be substantiallyfree of other cellular material and/or chemicals. A suitable anti-TNFαantibody is adalimumab.

As used herein, the term “adalimumab,” also known by its trade nameHUMIRA® (AbbVie) refers to a human IgG₁ antibody that binds human tumornecrosis factor α (TNFα). In general, the heavy chain constant domain 2(CH2) of the adalimumab IgG-Fc region is glycosylated through covalentattachment of oligosaccharide at asparagine 297 (Asn-297). The lightchain variable region of adalimumab is provided herein as SEQ ID NO:1,and the heavy chain variable region of adalimumab is provided herein asSEQ ID NO:2. Adalimumab comprises a light chain variable regioncomprising a CDR1 of SEQ ID NO:7, a CDR2 of SEQ ID NO:5, and a CDR3 ofSEQ ID NO:3. Adalimumab comprises a heavy chain variable regioncomprising a CDR1 of SEQ ID NO:8, a CDR2 of SEQ ID NO:6 and CDR3 of SEQID NO:4. The nucleic acid sequence of the light chain variable region isset forth in SEQ ID NO:9. The nucleic acid sequence of the heavy chainvariable region is set forth in SEQ ID NO:10. The full length amino acidsequence of the light chain is set forth as SEQ ID NO:11 and the fulllength amino acid sequence of the heavy chain is set forth as SEQ IDNO:12. Adalimumab is described in U.S. Pat. Nos. 6,090,382; 6,258,562;6,509,015; 7,223,394; 7,541,031; 7,588,761; 7,863,426; 7,919,264;8,197,813; 8,206,714; 8,216,583; 8,420,081; 8,092,998; 8,093,045;8,187,836; 8,372,400; 8,034,906; 8,436,149; 8,231,876; 8,414,894;8,372,401, the entire contents of each which are expressly incorporatedherein by reference in their entireties. Adalimumab is also described inthe “Highlights of Prescribing Information” for HUMIRA® (adalimumab)Injection (Revised January 2008) the contents of which are herebyincorporated herein by reference.

As used herein, a heavy chain antigen binding domain (referred to hereinas VD or VDH) is intended to include a heavy chain variable domain, adual heavy chain variable domain, a triple heavy chain variable domain,a light chain variable domain, a dual light chain variable domain, atriple light chain variable domain, a heavy chain variable domain incombination with a light chain variable domain, two heavy chain variabledomains in combination with a light chain variable domain, a heavy chainvariable domain in combination with two light chain variable domains, adomain antibody, a camelid antibody, a scFv, a receptor, and a scaffoldantigen binding protein. It is understood that the heavy chain antigenbinding domain may or may not bind an antigen independently of a pairedlight chain, dual light chain, or triple light chain, as appropriate,present on a second polypeptide of the binding proteins of theinvention. For example, a domain antibody, a scFv, or a receptor wouldbe expected to bind a target independent of any amino acid sequences ona second polypeptide claim. As the binding proteins of the inventionform functional antigen binding sites, if the heavy chain antigenbinding domain cannot specifically bind a target antigen independently(i.e., does not alone provide a functional antibody binding site), asecond polypeptide should be present to provide a complementary lightchain variable domain to provide a functional antibody binding site.

As used herein, a light chain antigen binding domain (referred to hereinas VD or VDL) is intended to include a light chain variable domain, adual light chain variable domain, a triple light chain variable domain,a heavy chain variable domain, a dual heavy chain variable domain, atriple heavy chain variable domain, a heavy chain variable domain incombination with a light chain variable domain, two heavy chain variabledomains in combination with a light chain variable domain, a heavy chainvariable domain in combination with two light chain variable domains, acamelid antibody, a domain antibody, a camelid antibody, a scFv, areceptor, and a scaffold antigen binding protein. It is understood thatthe light chain antigen binding domain may or may not bind an antigenindependently of a paired heavy chain, dual heavy chain, or triple heavychain, as appropriate, present on another polypeptide of the bindingproteins of the invention. For example, a domain antibody, a scFv, or areceptor would be expected to bind a target independent of any aminoacid sequences on a second polypeptide claim.

As used herein, “VD” alone can be understood to be either a heavy chainantigen binding domain or a light chain antigen binding domain unlessotherwise clear from context.

As used herein, “Dual Variable Domain Immunoglobulin” or “DVD-Ig™,” andthe like are understood to include binding proteins having the structureschematically represented in FIG. 19 and provided in US PatentPublications 20100260668 and 20090304693 both of which are incorporatedherein by reference. DVDs may be monospecific, i.e., bind one antigen,or multispecific, i.e. bind two or more antigens. A DVD-Ig™ comprises apaired heavy chain DVD polypeptide and a light chain DVD polypeptidewith each paired heavy and light chain providing two antigen bindingsites. Each binding site includes a total of 6 CDRs involved in antigenbinding per antigen binding site. A DVD-Ig™ is typically has two armsbound to each other at least in part by dimerization of the CH3 domains,with each arm of the DVD being bispecific, providing an immunoglobulinwith four binding sites.

A TVD-Ig is described in PCT Publication No. WO 2012/088290, the entirecontents of which are incorporated herein by reference. A half-body isdescribed in PCT Publication No. WO 2012/088302, the entire contents ofwhich are incorporated herein by reference.

As used herein, the term “upstream process technology,” in the contextof protein, e.g., antibody, preparation, refers to activities involvingthe production and collection of proteins (e.g. antibodies or DVD-Igs)from cells (e.g., during cell culture of a protein with a modulatedglycosylation profile). As used herein, the term “cell culture” refersto methods and techniques employed to generate and maintain a populationof host cells capable of producing a recombinant protein with amodulated glycosylation profile, as well as the methods and techniquesfor optimizing the production and collection of the protein with amodulated glycosylation profile. For example, once an expression vectorhas been incorporated into an appropriate host, the host can bemaintained under conditions suitable for high level expression of therelevant nucleotide coding sequences, and the collection andpurification of the desired recombinant protein.

When using the cell culture techniques of the instant invention, theprotein with a modulated glycosylation profile can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. In embodiments where the protein with a modulated glycosylationprofile is produced intracellularly, the particulate debris, either hostcells or lysed cells (e.g., resulting from homogenization), can beremoved by a variety of means, including but not limited to, bycentrifugation or ultrafiltration. Where the protein with a modulatedglycosylation profile is secreted into the medium, supernatants fromsuch expression systems can be first concentrated using a commerciallyavailable protein concentration filter, e.g., an Amicon™ or MilliporePellicon™ ultrafiltration unit

As used herein, the term “downstream process technology” refers to oneor more techniques used after the upstream process technologies topurify the protein, e.g., antibody, antigen-binding portion thereof, orDVD-Ig, of interest. For example, downstream process technology includespurification of the protein product, using, for example, affinitychromatography, including Protein A affinity chromatography, ionexchange chromatography, such as anion or cation exchangechromatography, hydrophobic interaction chromatography, displacementchromatography, multi-mode chromatography, continuous and recyclechromatography, viral filtration, depth filtration, ultrafiltration,diafiltration and centrifugation.

As used herein a “recombinant expression vector” can be any suitablerecombinant expression vector, and can be used to transform or transfectany suitable host. For example, one of ordinary skill in the art wouldappreciate that transformation or transfection is a process by whichexogenous nucleic acid such as DNA is introduced into a cell wherein thetransformation or transfection process involves contacting the cell withthe exogenous nucleic acid such as the recombinant expression vector asdescribed herein. Non-limiting examples of such expression vectors arethe pUC series of vectors (Fermentas Life Sciences), the pBluescriptseries of vectors (Stratagene, LaJolla, Calif.), the pET series ofvectors (Novagen, Madison, Wis.), the pGEX series of vectors (PharmaciaBiotech, Uppsala, Sweden), and the pEX series vectors (Clontech, PaloAlto, Calif.).

The phrase “recombinant host cell” (or simply “host cell”) includes acell into which a recombinant expression vector has been introduced. Itshould be understood that such terms are intended to refer not only tothe particular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein. In an embodiment, host cellsinclude prokaryotic and eukaryotic cells selected from any of theKingdoms of life. In another embodiment, eukaryotic cells includeprotist, fungal, plant and animal cells. In another embodiment, hostcells include, but are not limited to, the prokaryotic cell line E.coli; mammalian cell lines CHO, HEK 293, COS, NS0, SP2 and PER.C6; theinsect cell line Sf9; and the fungal cell Saccharomyces cerevisiae.

In certain embodiments, the host cells used in the methods of thepresent invention are prokaryote, yeast, or higher eukaryote cells.Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, e.g., Enterobacteriaceae suchas Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella,Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g.,Serratia marcescans, and Shigella, as well as Bacilli such as B.subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed inDD 266,710 published Apr. 12, 1989), Pseudomonas such as P. aeruginosa,and Streptomyces. One suitable E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X1776 (ATCC31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examplesare illustrative rather than limiting.

In certain embodiments, the host cells are eukaryotic microbes such asfilamentous fungi or yeast. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

In certain embodiments the host cells are derived from multicellularorganisms. In particular embodiments, the cells are invertebrate cellsfrom plant and insect cells. Non-limiting examples include cells derivedfrom Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito),Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), Bombyxmori, cotton, corn, potato, soybean, petunia, tomato, and tobacco canalso be utilized. As used herein, the term “recombinant protein” refersto a protein produced as the result of the transcription and translationof a gene carried on a recombinant expression vector that has beenintroduced into a host cell. In certain embodiments the recombinantprotein is an antibody, preferably a chimeric, humanized, or fully humanantibody. In certain embodiments the recombinant protein is an antibodyof an isotype selected from group consisting of: IgG (e.g., IgG1, IgG2,IgG3, IgG4), IgM, IgA1, IgA2, IgD, or IgE. In certain embodiments theantibody molecule is a full-length antibody (e.g., an IgG1 or IgG4immunoglobulin) or alternatively the antibody can be a fragment (e.g.,an Fc fragment or a Fab fragment). In some embodiments, the recombinantprotein is a DVD-Ig, a TVD-Ig, a RAB or a half-body.

In the methods of the invention, the host cells are cultured in mediasupplemented with an oligosaccharide and/or a monosaccharide. As usedherein, the term “monosaccharide” refers to any of a class ofcarbohydrates that cannot be broken down to simpler sugars by hydrolysisand that constitute the building blocks of oligosaccharides andpolysaccharides. Monosaccharides consist of at least three carbon atoms,one of which is attached to an oxygen atom to form an aldehyde group(CHO) or a ketone, and the others of which are each attached to ahydroxyl group (OH). A monosaccharide comprising three carbons permolecule is referred to a triose. A monosaccharide comprising fourcarbons per molecule is referred to as a tetrose. A monosaccharidecomprising five carbons per molecule is referred to as a pentose. Amonosaccharide sugar containing six carbons per molecule is referred toas a hexose. Monosaccharides can occur as chains or rings. Non-limitingexamples of monosaccharides include tagatose, glucose, galactose,ribose, fructose and xylose. In one embodiment of the invention, themonosaccharide is not glucose.

As used herein the term “oligosaccharide” refers to a saccharide polymercontaining a small number of, generally two to ten, monosaccharides. Themonosaccharide units are bonded to each other by glycosidic linkages.Non-limiting examples of oligosaccharides include sucrose, lactose,maltose, raffinose, trehalose, melibiose, maltotriose, gentianose andmaltopentalose.

The term “about”, as used herein, is intended to refer to ranges ofapproximately 0.1-2.0% greater than or less than the referenced value.In certain circumstances, one of skill in the art will recognize that,due to the nature of the referenced value, the term “about” can meanmore or less than a 0.1-2.0% deviation from that value.

The term “control”, as used herein, is intended to refer to acomposition comprising a protein produced by culturing a host cellexpressing a protein in cell culture media which is not supplementedwith a monosaccharide and/or an oligosaccharide. For example, a controlmay include a composition comprising a protein (e.g., an antibody)produced using the same host cell line and the same recombinantexpression vector under the same cell culture conditions, including thesame culture media, same culture vessel, same culture mode, same culturetemperature and same pH, but without monosaccharide or oligosaccharidesupplementation. For example, if antibody X is the antibody whoseglycosylation profile is modulated using the methods of the invention,the control would be a composition comprising antibody X produced usingthe same host cell line and the same recombinant expression vector underthe same cell culture conditions, including the same culture media, sameculture vessel, same culture mode, same culture temperature and same pH,but without monosaccharide or oligosaccharide supplementation.

II. Modulation of Protein Glycosylation Using Monosaccharides andOligosaccharides Glycosylation

It is well known that the pattern of glycoforms that arise inrecombinant proteins, including monoclonal antibodies, can be affectedby culture conditions during production (Nam et al. (2008) Biotechnol.Bioeng. 100(6): 1178-92). Consistency in the quality of theglycoproteins is important as glycosylation may impact proteinsolubility, activity, and circulatory half-life. (Gawlitzek et al.(1995) Biotechnol. Bioeng. 46:536-544; and Hayter et al. (1992)Biotechnol. Bioeng. 39:327-335).

Post-translational modification of nascent recombinant proteins includesenzymatic glycosylation. The resulting proteins, bearing covalentlylinked oligosaccharide side chains, are known as glycosylated proteinsor glycoproteins. Antibodies are glycoproteins with one or morecarbohydrate residues in the Fc domain, as well as the variable domain.Carbohydrate residues in the Fc domain have an important effect on theeffector function of the Fc domain, with minimal effect on antigenbinding or half-life of the antibody (Jefferis, R. Biotechnol. Prog.(2005) 21:11-16). In contrast, glycosylation of the variable domain mayhave an effect on the antigen binding activity of the antibody.Glycosylation in the variable domain may also have a negative effect onantibody binding affinity, likely due to steric hindrance (Co, M. S. etal., (1993) Mol. Immunol. 30:1361-1367), or result in increased affinityfor the antigen (Wallick, S. C. et al., (1988) Exp. Med. 168:1099-1109;Wright, A. et al., (1991) EMBO J. 10:2717 2723).

Protein glycosylation depends on the amino acid sequence of the proteinof interest, as well as the host cell in which the protein is expressed.Different organisms may produce different glycosylation enzymes (e.g.,glycosyltransferases and glycosidases), and have different substrates(nucleotide sugars) available. Due to such factors, proteinglycosylation pattern, and composition of glycosyl residues, may differdepending on the host system in which the particular protein isexpressed. Glycosyl residues useful in the proteins produced using themethods of the present invention may include, but are not limited to,glucose, galactose, mannose, fucose, n-acetylglucosamine, NGA2F-GlcNAc,NGA2F, NA1F-GlcNAc, NA1F, NA2F and sialic acid.

In one aspect of the present invention, the glycosylation of a protein,e.g., antibody, antigen-binding portion thereof, or DVD-Ig, ismodulated. Glycosylation can be modulated to, for example, increase theaffinity of the antibody or antigen-binding portion for the antigen.Such carbohydrate modifications can be accomplished by, for example,altering upstream process technologies, for example, recombinant hostcell culture conditions by supplementing the cell culture media with anoligosaccharide (e.g., sucrose) and/or a monosaccharide (e.g.,tagatose).

Additionally or alternatively, the present invention provides methodsfor modulating the glycosylation profile of a protein (e.g., an antibodyor DVD-Ig), such as modulating the type of glycan species and/or theamount or level of glycan species present in the protein. For example,the methods of the present invention can be used to produce ahypofucosylated antibody having decreased amounts or levels of fucosylresidues. Such altered glycosylation patterns have been demonstrated toincrease the ADCC ability of antibodies. In one embodiment, the amountof NGA2F species linked to the protein is decreased, for example, theamount or level of NGA2F species linked to the protein is decreased byabout 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%,4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,51%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.Ranges within one or more of the preceding values, e.g., about 0.1% to50%, 1% to 50%, 1% to 51%, 1% to 55%, 1% to 60%, 5% to 50%, 5% to 51%,5% to 55%, 5% to 60%, 9% to 51%, 10% to 60%, or 0.1% to 99% arecontemplated by the invention.

In another embodiment, the modulation of the glycosylation of theprotein results in an increase in the amount or level of NGA2F-GlcNAcspecies linked to the protein, for example, the amount or level ofNGA2F-GlcNAc species linked to the protein is increased by about 0.1%,1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 6%,7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or 99%. Ranges within one or more of thepreceding values, e.g., about 0.1% to 5%, 0.1% to 10%, 1% to 10%, 2% to8%, 3% to 6%, 5% to 8% or 0.1% to 99% are contemplated by the invention.

In another embodiment, the modulation of the glycosylation of theprotein results in an increase or a decrease in the amount or level ofNA1F-GlcNAc species linked to the protein, for example, the amount orlevel of NA1F-GlcNAc species linked to the protein is increased ordecreased by about 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%,4%, 4.2%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. Rangeswithin one or more of the preceding values, e.g., about 0.1-5%, 0.1-10%,1% to 10%, 2% to 8%, 3% to 6%, 5% to 8% or 0.1% to 99% are contemplatedby the invention.

In another embodiment, the modulation of the glycosylation of theprotein results in an increase or a decrease in the amount or level ofNA1F species linked to the protein, for example, the amount or level ofNA1F species linked to the protein is increased or decreased by about0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%,5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. Ranges within one or moreof the preceding values, e.g., about 0.1-5%, 0.1-10%, 1% to 10%, 2% to8%, 3% to 6%, 5% to 8% or 0.1% to 99% are contemplated by the invention.

In another embodiment, the modulation of the glycosylation of theprotein results in an increase or a decrease in the amount or level ofNA2F species linked to the protein, for example, the amount or level ofNA2F species linked to the protein is increased or decreased by about0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%,5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. Ranges within one or moreof the preceding values, e g., about 0.1-5%, 0.1-10%, 1% to 10%, 2% to8%, 3% to 6%, 5% to 8% or 0.1% to 99% are contemplated by the invention.

In another embodiment, the modulation of the glycosylation of theprotein results in an increase in the amount or level of NGA2F-GlcNAc,NA1F-GlcNAc, NA1F and/or NA2F species linked to the protein, for examplethe amount or level of NGA2F-GlcNAc, NA1F-GlcNAc, NA1F and/or NA2Flinked to the protein is increased by about 0.1%, 1%, 1.2%, 1.5%, 2%,2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or 99%. Ranges within one or more of the preceding values,e.g., about 0.1% to 5%, 0.1% to 10%, 0.1% to 20%, 1% to 10%, 1% to 20%,2% to 8%, 3% to 6%, 3% to 20%, 5% to 8%, 5% to 20% or 0.1% to 99%.

In another embodiment, the modulation of the glycosylation of theprotein results in a decrease in the amount or level of NGA2F,NA1F-GlcNAc, NA1F and/or NA2F species linked to the protein, for examplethe amount or level of NGA2F, NA1F-GlcNAc, NA1F and/or NA2F linked tothe protein is decreased by about 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%,3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,99%. Ranges within one or more of the preceding values, e.g., about 0.1%to 5%, 0.1% to 10%, 0.1% to 20%, 0.1% to 30%, 0.1% to 40%, 0.1% to 50%,0.1% to 60%, 0.1% to 65%, 1% to 10%, 1% to 20%, 1% to 30%, 1% to 40%, 1%to 50%, 1% to 60%, 1% to 65%, 5% to 10%, 5% to 20%, 5% to 30%, 5% to40%, 5% to 50%, 5% to 60%, 5% to 65%, 10% to 20%, 10% to 30%, 10% to40%, 10% to 50%, 10% to 60%, 10% to 65%, or 0.1 to 99%.

In another embodiment, the overall fucosylation level resulting from themodulation (e.g., increase or decrease) of any one of the fucosylatedglycan species such as NGA2F-GlcNAc, NGA2F, NA1F-GlcNAc, NA1F and/orNA2F is decreased by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%,3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. Ranges within one ormore of the preceding values, e.g., about 1-10%, 1-15%, 1-20%, 1-25%,1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%,1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%,2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%,3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%,3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-10%, 4-15%, 4-20%,4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%,4-47%, 4-48%, 4-49%, 4-50%, 5-10%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%,5-40%, 5-41%, 5-42%, 5-43, 5-44%, 5-45%, 5-46%, 5-47%, 5-48%, 5-49%,5-50% or 1-99% are contemplated by the invention.

In another embodiment, the modulation of the glycosylation of theprotein (e.g., antibody or DVD-Ig) results in an increase in the amountor level of mannosylation, for example, the amount or level ofmannosylation is increased by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%,3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99%. Ranges withinone or more of the preceding values, e.g., about 1-5%, 1-10%, 1-15%,1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%,1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-5%, 2-10%, 2-15%, 2-20%, 2-25%,2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%,2-48%, 2-49%, 2-50%, 3-5%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%,3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%,3-50%, 4-5%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%,4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50% or 1-99%are contemplated by the invention.

In another embodiment, the increase in mannosylation of the proteincomprises an increase in the amount or level of a high mannose N-glycanoligosaccharide. A high-mannose N-glycan has more than one mannoselinked to the non-reducing terminal of the core structure. For example,the high mannose N-glycan oligosaccharide is selected from the groupconsisting of Man 5 glycan, Man 6 glycan, Man 7 glycan and Man 8 glycan.In one embodiment, the amount or level of at least one of Man 5 glycan,Man 6 glycan, Man 7 glycan and/or Man 8 glycan is increased by about0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or 99%. Ranges within one or more of thepreceding values, e.g., about 0.1-5%, 0.1-10%, 0.1-15%, 0.1-20%,0.1-21%, 0.1-22%, 0.1-23%, 0.1-24%, 0.1-25%, 0.1-26%, 0.1-27%, 0.1-28%,0.1-29%, 0.1-30%, 0.1-35%, 0.1-40%, 0.1-41%, 0.1-42%, 0.1-43%, 0.1-44%,0.1-45%, 0.1-46%, 0.1-47%, 0.1-48%, 0.1-49%, 0.1-50%, 1-5%, 1-10%,1-15%, 1-20%, 1-21%, 1-22%, 1-23%, 1-24%, 1-25%, 1-26%, 1-27%, 1-28%,1-29%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%,1-47%, 1-48%, 1-49%, 1-50%, 2-5%, 2-10%, 2-15%, 2-20%, 2-21%, 2-22%,2-23%, 2-24%, 2-25%, 2-26%, 2-27%, 2-28%, 2-29%, 2-30%, 2-35%, 2-40%,2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%,3-5%, 3-10%, 3-15%, 3-20%, 3-21%, 3-22%, 3-24%, 3-25%, 3-26%, 3-27%,3-28%, 2-29%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%,3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-5%, 4-10%, 4-15%, 4-20%, 4-25%,4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%,4-48%, 4-49%, 4-50% or 0.1-99% are contemplated by the invention.

It is known to those skilled in the art that differing proteinglycosylation profiles may result in differing protein characteristics.For instance, the efficacy of a therapeutic protein produced in amicroorganism host, such as yeast, and glycosylated utilizing the yeastendogenous pathway may be reduced compared to that of the same proteinexpressed in a mammalian cell, such as a CHO cell line. Suchglycoproteins may also be immunogenic in humans and show reducedhalf-life in vivo after administration. Specific receptors in humans andother animals may recognize specific glycosyl residues and promote therapid clearance of the protein from the bloodstream. Other adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, using the methodsof the invention, one of skill in the art may modulate the glycosylationprofile of a protein, e.g., an antibody or DVD-Ig to achieve a desiredactivity such as increased or decreased rate of clearance and/orincreased ADCC activity.

Upstream Process Technologies

The methods of the present invention may be used to produce a protein(e.g., an antibody, or antigen binding fragment thereof, or a DVD-Ig)with a modulated glycosylation profile. In one embodiment, the methodsof the invention involve modification of the conditions used duringupstream protein production, such as recombinant cell cultureconditions. For example, the methods of the invention comprisesupplementing the recombinant cell culture media with a monosaccharide(e.g., tagatose) and/or oligosaccharide (e.g., sucrose) to modulate theglycosylation profile of the protein.

The upstream process technologies may be used alone or in combinationwith the downstream process technologies described below.

In one embodiment, the methods described herein produce a protein with amodulated glycosylation profile wherein the overall fucosylation levelresulting from the modulation of any one of the fucosylated glycanspecies such as NGA2F-GlcNAc, NGA2F, NA1F-GlcNAc, NA1F and/or NA2F, isdecreased by about 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%,4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, and ranges within one or more ofthe preceding. In one aspect of this embodiment, the overallfucosylation level is decreased by about 1-10%, 1-15%, 1-20%, 1-25%,1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%,1-48%, 1-49%, 1-50%, 2-10%, 2-15%, 2-20%, 2-25%, 2-30%, 2-35%, 2-40%,2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%,3-10%, 3-15%, 3-20%, 3-25%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%,3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%, 3-50% 4-10%, 4-15%, 4-20%,4-25%, 4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%,4-47%, 4-48%, 4-49%, 4-50%, 5-10%, 5-15%, 5-20%, 5-25%, 5-30%, 5-35%,5-40%, 5-41%, 5-42%, 5-43, 5-44%, 5-45%, 5-46%, 5-47%, 5-48%, 5-49%,5-50% or 1-99% and ranges within one or more of the preceding.

In another embodiment, the methods described herein produce a proteinwith a modulated glycosylation profile wherein the overall mannosylationlevel resulting from the modulation of any one of the high mannoseN-glycan oligosaccharides, such as Man 5 glycan, Man 6 glycan, Man 7glycan or Man 8 glycan, is increased by about 0.1%, 1%, 1.2%, 1.5%, 2%,2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%,and ranges within one or more of the preceding. In one aspect of thisembodiment, the overall high mannose N-glycan level is increased byabout 0.1-5%, 0.1-10%, 0.1-15%, 0.1-20%, 0.1-21%, 0.1-22%, 0.1-23%,0.1-24%, 0.1-25%, 0.1-26%, 0.1-27%, 0.1-28%, 0.1-29%, 0.1-30%, 0.1-35%,0.1-40%, 0.1-41%, 0.1-42%, 0.1-43%, 0.1-44%, 0.1-45%, 0.1-46%, 0.1-47%,0.1-48%, 0.1-49%, 0.1-50%, 1-5%, 1-10%, 1-15%, 1-20%, 1-21%, 1-22%,1-23%, 1-24%, 1-25%, 1-26%, 1-27%, 1-28%, 1-29%, 1-30%, 1-35%, 1-40%,1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%,2-5%, 2-10%, 2-15%, 2-20%, 2-21%, 2-22%, 2-23%, 2-24%, 2-25%, 2-26%,2-27%, 2-28%, 2-29%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%,2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%, 3-5%, 3-10%, 3-15%, 3-20%,3-21%, 3-22%, 3-24%, 3-25%, 3-26%, 3-27%, 3-28%, 2-29%, 3-30%, 3-35%,3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%, 3-49%,3-50%, 4-5%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%,4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50% or0.1-99%, and ranges within one or more of the preceding.

In certain embodiments, the methods described herein produce a protein,e.g., an antibody, with a modulated glycosylation profile wherein theantibody's antibody-dependent cellular cytotoxicity (ADCC) response isincreased by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, andranges within one or more of the preceding. In one aspect of thisembodiment, the antibody's ADCC response is increased by about 1-5%,1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-45%, 1-50%, 5-10%,5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 8-10%, 8-15%,8-20%, 8-25%, 8-30%, 8-35%, 8-40%, 8-45%, 8-50%, 10-15%, 10-20%, 10-25%,10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 20-25%, 20-30%, 20-35%, 20-40%,20-45%, 20-50% or 1-99%, and ranges within one or more of the preceding.

As described herein, the host cell culture conditions can be modified ascompared to conditions during production of the same protein withoutmodulation of the glycosylation profile. In one embodiment, a proteinwith a modulated glycosylation profile is produced by culturing cellsexpressing the antibody, or antigen binding fragment thereof, or DVD-Igin a cell culture media supplemented with an oligosaccharide (e.g.,sucrose) and/or a monosaccharide (e.g., tagatose).

To express a protein with a modulated glycosylation profile (e.g., anantibody, or antigen-binding fragment thereof, or DVD-Ig), DNAs encodingthe protein, such as DNAs encoding partial or full-length light andheavy chains in the case of antibodies, are inserted into one or moreexpression vector such that the genes are operatively linked totranscriptional and translational control sequences. (See, e.g., U.S.Pat. No. 6,090,382, the entire contents of which are incorporated hereinby reference.) In this context, the term “operatively linked” isintended to mean that a gene encoding the protein is ligated into avector such that transcriptional and translational control sequenceswithin the vector serve their intended function of regulating thetranscription and translation of the gene. The expression vector andexpression control sequences are chosen to be compatible with theexpression host cell used. In certain embodiments, the protein with amodulated glycosylation profile will comprising multiple polypeptides,such as the heavy and light chains of an antibody. Thus, in certainembodiments, genes encoding multiple polypeptides, such as antibodylight chain genes and antibody heavy chain genes, can be inserted into aseparate vector or, more typically, the genes are inserted into the sameexpression vector. Genes are inserted into expression vectors bystandard methods (e.g., ligation of complementary restriction sites onthe gene fragment and vector, or blunt end ligation if no restrictionsites are present). Prior to insertion of the gene or genes, theexpression vector may already carry additional polypeptide sequences,such as, but not limited to, antibody constant region sequences. Forexample, one approach to converting the anti-TNFα antibody or anti-TNFαantibody-related VH and VL sequences to full-length antibody genes is toinsert them into expression vectors already encoding heavy chainconstant and light chain constant regions, respectively, such that theVH segment is operatively linked to the CH segment(s) within the vectorand the VL segment is operatively linked to the CL segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the proteinfrom a host cell. The gene can be cloned into the vector such that thesignal peptide is linked in-frame to the amino terminus of the gene. Thesignal peptide can be an immunoglobulin signal peptide or a heterologoussignal peptide (i.e., a signal peptide from a non-immunoglobulinprotein).

In addition to protein coding genes, a recombinant expression vector cancarry one or more regulatory sequence that controls the expression ofthe protein coding genes in a host cell. The term “regulatory sequence”is intended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals) that control the transcriptionor translation of the protein coding genes. Such regulatory sequencesare described, e.g., in Goeddel; Gene Expression Technology: Methods inEnzymology 185, Academic Press, San Diego, Calif. (1990), the entireteaching of which is incorporated herein by reference. It will beappreciated by those skilled in the art that the design of theexpression vector, including the selection of regulatory sequences maydepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, etc. Suitable regulatorysequences for mammalian host cell expression include viral elements thatdirect high levels of protein expression in mammalian cells, such aspromoters and/or enhancers derived from cytomegalovirus (CMV) (such asthe CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus, (e.g., the adenovirus major latepromoter (AdMLP)) and polyoma. For further description of viralregulatory elements, and sequences thereof, see, e.g., U.S. Pat. No.5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and U.S.Pat. No. 4,968,615 by Schaffner et al., the entire teachings of whichare incorporated herein by reference.

A recombinant expression vector may also carry one or more additionalsequences, such as a sequence that regulates replication of the vectorin host cells (e.g., origins of replication) and/or a selectable markergene. The selectable marker gene facilitates selection of host cellsinto which the vector has been introduced (see e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al., the entireteachings of which are incorporated herein by reference). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Suitable selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr-host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

An antibody, or antigen binding fragment thereof, to be used in themethod of preparing a protein with a modulated glycosylation profile canbe prepared by recombinant expression of immunoglobulin light and heavychain genes in a host cell. To express an antibody recombinantly, a hostcell is transfected with one or more recombinant expression vectorscarrying DNA fragments encoding the immunoglobulin light and heavychains of the antibody such that the light and heavy chains areexpressed in the host cell and secreted into the medium in which thehost cells are cultured, from which medium the antibodies can berecovered. Standard recombinant DNA methodologies are used to obtainantibody heavy and light chain genes, incorporate these genes intorecombinant expression vectors and introduce the vectors into hostcells, such as those described in Sambrook, Fritsch and Maniatis (eds),Molecular Cloning; A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), Ausubel et al. (eds.) Current Protocols inMolecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat.Nos. 4,816,397 & 6,914,128, the entire teachings of which areincorporated herein.

For expression of a protein, for example, the light and heavy chains ofan antibody, the expression vector(s) encoding the protein is (are)transfected into a host cell by standard techniques. The various formsof the term “transfection” are intended to encompass a wide variety oftechniques commonly used for the introduction of exogenous DNA into aprokaryotic or eukaryotic host cell, e.g., electroporation,calcium-phosphate precipitation, DEAE-dextran transfection and the like.Although it is theoretically possible to express the proteins of theinvention in either prokaryotic or eukaryotic host cells, expression ofantibodies in eukaryotic cells, such as mammalian host cells, issuitable because such eukaryotic cells, and in particular mammaliancells, are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active protein. Prokaryoticexpression of protein genes has been reported to be ineffective forproduction of high yields of active protein (Boss and Wood (1985)Immunology Today 6:12-13, the entire teaching of which is incorporatedherein by reference).

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, e.g., Enterobacteriaceae suchas Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella,Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g.,Serratia marcescans, and Shigella, as well as Bacilli such as B.subtilis and B. licheniformis (e.g., B. licheniformis 41P disclosed inDD 266,710 published Apr. 12, 1989), Pseudomonas such as P. aeruginosa,and Streptomyces. One suitable E. coli cloning host is E. coli 294 (ATCC31,446), although other strains such as E. coli B, E. coli X1776 (ATCC31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examplesare illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for polypeptideencoding vectors. Saccharomyces cerevisiae, or common baker's yeast, isthe most commonly used among lower eukaryotic host microorganisms.However, a number of other genera, species, and strains are commonlyavailable and useful herein, such as Schizosaccharomyces pombe;Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC 12,424),K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii(ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K.marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida;Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces suchas Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A.nidulans and A. niger.

Suitable host cells for the expression of proteins with modulatedglycosylation profiles, for example, glycosylated antibodies, arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells. Plant cell cultures ofcotton, corn, potato, soybean, petunia, tomato, and tobacco can also beutilized as hosts.

Mammalian cells can be used for expression and production of the proteincompositions of the invention, however other eukaryotic cell types canalso be employed in the context of the instant invention. See, e.g.,Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y. (1987).Suitable mammalian host cells for expressing recombinant proteinsaccording to the invention include Chinese Hamster Ovary (CHO cells)(including dhfr-CHO cells, described in Urlaub and Chasin, (1980) PNASUSA 77:4216-4220, used with a DHFR selectable marker, e.g., as describedin Kaufman and Sharp (1982) Mol. Biol. 159:601-621, the entire teachingsof which are incorporated herein by reference), NS0 myeloma cells, COScells and SP2 cells. When recombinant expression vectors encodingprotein genes are introduced into mammalian host cells, the antibodiesare produced by culturing the host cells for a period of time sufficientto allow for expression of the antibody in the host cells or secretionof the antibody into the culture medium in which the host cells aregrown. Other examples of useful mammalian host cell lines are monkeykidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); humanembryonic kidney line (293 or 293 cells subcloned for growth insuspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); babyhamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovarycells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216(1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2), the entire teachings of which are incorporated herein byreference.

Host cells are transformed with the above-described expression orcloning vectors for protein production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

The host cells used to produce a protein may be cultured in a variety ofmedia which are supplemented in accordance with the present invention.Commercially available media such as Ham's F10™ (Sigma), MinimalEssential Medium™ (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco'sModified Eagle's Medium™ (DMEM), (Sigma), Iscove's Modified Dulbecco'sMedium, Minimal Essential Medium-alpha. (MEM-alpha), DME/F12, alpha MEM,Basal Medium Eagle with Earle's BSS, DMEM high Glucose, withL-Glutamine, DMEM high glucose, without L-Glutamine, DMEM low Glucose,without L-Glutamine, DMEM:F12 1:1, with L-Glutamine, GMEM (Glasgow'sMEM), GMEM with L-glutamine, Grace's Complete Insect Medium, Grace'sInsect Medium, without FBS, Ham's F-10, with L-Glutamine, Ham's F-12,with L-Glutamine, IMDM with HEPES and L-Glutamine, IMDM with HEPES andwithout L-Glutamine, IPL-41 Insect Medium, L-15 (Leibovitz)(2.times.),without L-Glutamine or Phenol Red, L-15 (Leibovitz), withoutL-Glutamine, McCoy's 5A Modified Medium, Medium 199, MEM Eagle, withoutL-Glutamine or Phenol Red (2.times.), MEM Eagle-Earle's BSS, withL-glutamine, MEM Eagle-Earle's BSS, without L-Glutamine, MEM Eagle-HanksBSS, without L-Glutamine, NCTC-109, with L-Glutamine, Richter's CMMedium, with L-Glutamine, RPMI 1640 with HEPES, L-Glutamine and/orPenicillin-Streptomycin, RPMI 1640, with L-Glutamine, RPMI 1640, withoutL-Glutamine, Schneider's Insect Medium are suitable for culturing hostcells. In addition, any of the media described in Ham et al., Meth. Enz.58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat.Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used asculture media for the host cells, the entire teachings of which areincorporated herein by reference.

Any of these media may be supplemented as necessary with hormones and/orother growth factors (such as insulin, transferrin, or epidermal growthfactor), salts (such as sodium chloride, calcium, magnesium, andphosphate), buffers (such as HEPES), nucleotides (such as adenosine andthymidine), antibiotics (such as gentamycin drug), trace elements(defined as inorganic compounds usually present at final concentrationsin the micromolar range), and glucose or an equivalent energy source.Any other necessary supplements may also be included at appropriateconcentrations that would be known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are thosepreviously used with the host cell selected for expression, and will beapparent to the ordinarily skilled artisan.

Host cells can also be used to produce portions of intact proteins, forexample, antibodies, including Fab fragments or scFv molecules. It isunderstood that variations on the above procedure are within the scopeof the present invention. For example, in certain embodiments it may bedesirable to transfect a host cell with DNA encoding either the lightchain or the heavy chain (but not both) of an antibody. Recombinant DNAtechnology may also be used to remove some or all of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to an antigen. The molecules expressed from such truncated DNAmolecules are also encompassed by the antibodies of the invention. Inaddition, bifunctional antibodies may be produced in which one heavy andone light chain are an antibody of the invention and the other heavy andlight chain are specific for an antigen other than the target antibody,depending on the specificity of the antibody of the invention, bycrosslinking an antibody of the invention to a second antibody bystandard chemical crosslinking methods.

In a suitable system for recombinant expression of a protein, forexample, an antibody, or antigen-binding portion thereof, or a DVD-Ig, arecombinant expression vector encoding the protein, for example, both anantibody heavy chain and an antibody light chain, is introduced intodhfr-CHO cells by calcium phosphate-mediated transfection. Within therecombinant expression vector, the protein gene(s) are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the gene(s). The recombinant expressionvector also carries a DHFR gene, which allows for selection of CHO cellsthat have been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the protein, for example, theantibody heavy and light chains, and intact protein, for example, anantibody, is recovered from the culture medium. Standard molecularbiology techniques are used to prepare the recombinant expressionvector, transfect the host cells, select for transformants, culture thehost cells and recover the protein from the culture medium.

When using recombinant techniques, the protein, for example, antibodiesor antigen binding fragments thereof, can be produced intracellularly,in the periplasmic space, or directly secreted into the medium. In oneaspect, if the protein is produced intracellularly, as a first step, theparticulate debris, either host cells or lysed cells (e.g., resultingfrom homogenization), can be removed, e.g., by centrifugation orultrafiltration. Where the protein is secreted into the medium,supernatants from such expression systems can be first concentratedusing a commercially available protein concentration filter, e.g., anAmicon™ or Millipore Pellicon™ ultrafiltration unit.

Some antibodies can be secreted directly from the cell into thesurrounding growth media; others are made intracellularly. Forantibodies made intracellularly, the first step of a purificationprocess typically involves: lysis of the cell, which can be done by avariety of methods, including mechanical shear, osmotic shock, orenzymatic treatments. Such disruption releases the entire contents ofthe cell into the homogenate, and in addition produces subcellularfragments that are difficult to remove due to their small size. Theseare generally removed by differential centrifugation or by filtration.Where the antibody is secreted, supernatants from such expressionsystems are generally first concentrated using a commercially availableprotein concentration filter, e.g., an Amicon™ or Millipore Pellicon™ultrafiltration unit. Where the antibody is secreted into the medium,the recombinant host cells can also be separated from the cell culturemedium, e.g., by tangential flow filtration. Antibodies can be furtherrecovered from the culture medium using the antibody purificationmethods of the invention.

In accordance with the present invention, modulation of theglycosylation profile of the protein (e.g., antibody or DVD-Ig) producedby recombinant cell culture can be achieved by supplementation of thecell culture media with sucrose and/or tagatose. Specific host cellculture conditions can be used with various cultivation methodsincluding, but not limited to, batch, fed-batch, chemostat andperfusion, and with various cell culture equipment including, but notlimited to, shake flasks with or without suitable agitation, spinnerflasks, stirred bioreactors, airlift bioreactors, membrane bioreactors,reactors with cells retained on a solid support or immobilized/entrappedas in microporous beads, and any other configuration appropriate foroptimal growth and productivity of the desired host cell line.

Supplementation with Monosaccharides and/or Oligosaccharides to Modulatethe Glycosylation Profile of the Expressed Protein

The present invention relates to modulation of the glycosylation profilein mammalian cell culture processes using cell culture component such asmonosaccharide and/or oligosaccharide supplementation. These nutrientsare also important for ensuring both robust cell growth and productionof glycoproteins. In the present invention these components are utilizedto affect the profile of glycosylation of the glycoprotein. For example,but not by way of limitation, by adjusting the concentration of one orboth of these sugars the glycosylation profile can be modulated. Thus,the present invention provides methods to modulate the glycosylationprofile introduced by upstream process technologies to achieve desiredproduct glycosylation profiles.

In certain embodiments, a protein with a modulated glycosylation profileis prepared by supplementation of cell culture media withmonosaccharides (e.g., tagatose) and/or oligosaccharides (e.g.,sucrose). For example, supplementation with sucrose and/or tagatoseresults in a significant increase in non-fully processed N-glycans,including high mannose N-glycan species. This is consistent with theabrogation of the N-glycan biosynthetic pathway at the enzymaticreaction steps shown in FIG. 2, which results in the accumulation ofthese particular N-glycans. For some particular recombinantglycoproteins, a high mannose isoform is a desired product qualityattribute (Walsh, G. et al., (2006) Nat. Biotechnol. 24(10):1241-52). Inaddition, as a result of the increase in mannosylation, the levels offucosylation are significantly decreased. The addition of fucose toN-glycans has been shown to reduce antibody dependent cellularcytotoxicity (ADCC) (Kanda Y. et al., (2007) Glycobiology, 17(1):104-18;Shields R. L., et al., (2002) J. Biol. Chem. 2002. 277(30):26733-26740). Thus, where the ADCC response is the principle therapeuticmechanism of antibody activity, the provision of methods for thepreparation of recombinant protein therapeutics with a glycosylationprofile characterized by decreased fuscosylation, are beneficial.

In certain embodiments, the cell culture media is supplemented with oneor more monosaccharides or oligosaccharides in order to modulate theglycosylation profile of the protein (e.g., an antibody, of antigenbinding fragment thereof, or a DVD-Ig). In one embodiment themonosaccharide is tagatose. In one embodiment the cell culture media issupplemented with about 1 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 15mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM or 100 mMtagatose. In particular embodiments, the cell culture media issupplemented with about 1 mM, 10 mM, 30 mM, 50 mM or 70 mM tagatose.

In another embodiment the oligosaccharide is sucrose. In one embodimentthe cell culture media is supplemented with about 1 mM, 5 mM, 6 mM, 7mM, 8 mM, 9 mM, 10 mM, 15 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM,80 mM, 90 mM or 100 mM sucrose. In particular embodiments, the cellculture media is supplemented with about 1 mM, 7 mM, 10 mM, 15 mM, 30mM, 50 mM or 70 mM sucrose.

In certain embodiments, the cell culture media is supplemented with oneor more monosaccharides or oligosaccharides in an amount effective tomodulate the glycosylation profile of the protein such that the overallfucosylation amount or level, resulting from the modulation of at leastone of the fucosylated glycan species such as NGA2F-GlcNAc, NGA2F,NA1F-GlcNAc, NA1F and/or NA2F, is decreased by about 1%, 1.2%, 1.5%, 2%,2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%,and ranges within one or more of the preceding. In one aspect of thisembodiment, the overall fucosylation amount or level is decreased byabout 1-10%, 1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%,1-43%, 1-44%, 1-45%, 1-46%, 1-47%, 1-48%, 1-49%, 1-50%, 2-10%, 2-15%,2-20%, 2-25%, 2-30%, 2-35%, 2-40%, 2-41%, 2-42%, 2-43%, 2-44%, 2-45%,2-46%, 2-47%, 2-48%, 2-49%, 2-50%, 3-10%, 3-15%, 3-20%, 3-25%, 3-30%,3-35%, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%, 3-46%, 3-47%, 3-48%,3-49%, 3-50%, 4-10%, 4-15%, 4-20%, 4-25%, 4-30%, 4-35%, 4-40%, 4-41%,4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%, 4-48%, 4-49%, 4-50%, 5-10%,5-15%, 5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-41%, 5-42%, 5-43, 5-44%,5-45%, 5-46%, 5-47%, 5-48%, 5-49%, 5-50% or 1-99%, and ranges within oneor more of the preceding.

In certain embodiments, the cell culture media is supplemented with oneor more monosaccharides or oligosaccharides in an amount effective tomodulate the glycosylation profile of the protein such that the overallmannosylation amount or level, resulting from the modulation of at leastone of the high mannose N-glycan oligosaccharides, such as Man 5 glycan,Man 6 glycan, Man 7 glycan or Man 8 glycan, is increased by about 0.1%,1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99%, and ranges within one or more of the preceding. Inone aspect of this embodiment, the overall high mannose N-glycan amountor level is increased by about 0.1-5%, 0.1-10%, 0.1-15%, 0.1-20%,0.1-21%, 0.1-22%, 0.1-23%, 0.1-24%, 0.1-25%, 0.1-26%, 0.1-27%, 0.1-28%,0.1-29%, 0.1-30%, 0.1-35%, 0.1-40%, 0.1-41%, 0.1-42%, 0.1-43%, 0.1-44%,0.1-45%, 0.1-46%, 0.1-47%, 0.1-48%, 0.1-49%, 0.1-50%, 1-5%, 1-10%,1-15%, 1-20%, 1-21%, 1-22%, 1-23%, 1-24%, 1-25%, 1-26%, 1-27%, 1-28%,1-29%, 1-30%, 1-35%, 1-40%, 1-41%, 1-42%, 1-43%, 1-44%, 1-45%, 1-46%,1-47%, 1-48%, 1-49%, 1-50%, 2-5%, 2-10%, 2-15%, 2-20%, 2-21%, 2-22%,2-23%, 2-24%, 2-25%, 2-26%, 2-27%, 2-28%, 2-29%, 2-30%, 2-35%, 2-40%,2-41%, 2-42%, 2-43%, 2-44%, 2-45%, 2-46%, 2-47%, 2-48%, 2-49%, 2-50%,3-5%, 3-10%, 3-15%, 3-20%, 3-21%, 3-22%, 3-24%, 3-25%, 3-26%, 3-27%,3-28%, 2-29%, 3-30%, 3-35%, 3-40%, 3-41%, 3-42%, 3-43%, 3-44%, 3-45%,3-46%, 3-47%, 3-48%, 3-49%, 3-50%, 4-5%, 4-10%, 4-15%, 4-20%, 4-25%,4-30%, 4-35%, 4-40%, 4-41%, 4-42%, 4-43%, 4-44%, 4-45%, 4-46%, 4-47%,4-48%, 4-49%, 4-50% or 0.1-99%, and ranges within one or more of thepreceding.

In certain embodiments, the cell culture media is supplemented with oneor more monosaccharides or oligosaccharides in an amount effective tomodulate the glycosylation profile of the protein, e.g., an antibody,such that the antibody's antibody-dependent cellular cytotoxicity (ADCC)response is increased by about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,99%, and ranges within one or more of the preceding. In one aspect ofthis embodiment, the antibody's ADCC response is increased by about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or by about 1-5%, 1-10%,1-15%, 1-20%, 1-25%, 1-30%, 1-35%, 1-40%, 1-45%, 1-50%, 5-10%, 5-15%,5-20%, 5-25%, 5-30%, 5-35%, 5-40%, 5-45%, 5-50%, 8-10%, 8-15%, 8-20%,8-25%, 8-30%, 8-35%, 8-40%, 8-45%, 8-50%, 10-15%, 10-20%, 10-25%,10-30%, 10-35%, 10-40%, 10-45%, 10-50%, 20-25%, 20-30%, 20-35%, 20-40%,20-45%, 20-50% or 1-99%, and ranges within one or more of the preceding.

In certain embodiments, the cell culture media is supplemented, forexample, at the start of culture, or in a fed-batch or in a continuousmanner. The feed amounts may be calculated to achieve a certainconcentration based on off-line or on-line measurements. The addition ofone or more supplements may be based on measured glycosylation profiles.The resulting media can be used in various cultivation methodsincluding, but not limited to, batch, fed-batch, chemostat andperfusion, and with various cell culture equipment including, but notlimited to, shake flasks with or without suitable agitation, spinnerflasks, stirred bioreactors, airlift bioreactors, membrane bioreactors,reactors with cells retained on a solid support or immobilized/entrappedas in microporous beads, incubation vessels, microtiter plates,capillaries, multi-well plates and any other configuration appropriatefor optimal growth and productivity of the desired host cell line.Additional cell culture equipment may be used such as fermentor tanks,air lifts, culture flasks, spinner flasks, microcarriers, fluidizedbeds, hollow fibers, roller bottles or packed beds. In addition, theharvest criterion for these cultures may be chosen, for example, basedon choice of harvest viability or culture duration, to further optimizea certain targeted glycosylation profiles.

Down Stream Process Technologies

The protein compositions of the invention may be purified usingdownstream process technologies (e.g., purification or concentration),following production using the upstream process technologies of thepresent invention. For example, once a clarified solution or mixturecomprising the protein with a modulated glycosylation profile, e.g., anantibody or DVD-Ig, has been obtained, separation of the protein fromprocess-related impurities, such as the other proteins produced by thehost cell, as well as product-related substances, such acidic or basicvariants, is performed. In certain embodiments, the initial steps of thepurification methods involve the clarification and primary recovery ofan antibody or DVD-Ig from a sample matrix by methods such ascentrifugation, depth filtration and/or viral inactivation/reduction. Incertain non-limiting embodiments, further separation is performed usingcation exchange chromatography, anion exchange chromatography, and/ormulti-mode chromatography. In certain embodiments, a combination of oneor more different purification techniques, including affinity separationstep(s), ion exchange separation step(s), mixed-mode step(s), and/orhydrophobic interaction separation step(s) can also be employed. Suchadditional purification steps separate mixtures of proteins on the basisof their charge, degree of hydrophobicity, and/or size. Continuous andrecycle chromatography are also applicable to chromatography methodswhere the protein with a modulated glycosylation profile is collected inthe unbound faction during chromatography or where the protein is firstbound to the chromatography resin and subsequently recovered by washingthe media with conditions that elute the bound component. Numerouschromatography resins are commercially available for each of thesetechniques, allowing accurate tailoring of the purification scheme tothe particular protein involved. Each of the separation methods allowproteins to either traverse at different rates through a column,achieving a physical separation that increases as they pass furtherthrough the column, or to adhere selectively to a separation resin (ormedium). The proteins are then differentially eluted using differenteluents. In some cases, the protein with a modulated glycosylationprofile is separated from impurities when the impurities specificallyadhere to the column's resin and the protein does not, i.e., the proteinis contained in the effluent, while in other cases the protein willadhere to the column's resin, while the impurities and/orproduct-related substances are extruded from the column's resin during awash cycle. Following chromatographic polishing steps the proteincompositions of the invention may be further purified using viralfiltration. Ultrafiltration and/or diafiltration may be used to furtherconcentrate and formulate the protein, e.g., an antibody or DVD-Igproduct.

The glycosylation profile of the protein prepared by the methods of theinvention can be analyzed using methods well known to those skilled inthe art, e.g., removal and derivatization of N-glycans followed byNP-HPLC analysis, weak cation exchange chromatography (WCX), capillaryisoelectric focusing (cIEF), size-exclusion chromatography, Poros A HPLCAssay, Host cell Protein ELISA, DNA assay, and western blot analysis.

III. Methods of Treatment Using Proteins with Modulated GlycosylationProfiles of the Invention

The compositions comprising a protein with a modulated glycosylationprofile, for example a protein such as an antibody, antigen-bindingportion thereof, or a DVD-Ig, with a decreased fucosylation level oramount and/or an increased mannosylation level or amount, of theinvention may be used to treat any disorder in a subject for which thetherapeutic protein (e.g., an antibody, or an antigen binding fragmentthereof, or a DVD-Ig) comprised in the composition is appropriate fortreating.

A “disorder” is any condition that would benefit from treatment with thetherapeutic protein with a modulated glycosylation profile. Thisincludes chronic and acute disorders or diseases including thosepathological conditions which predispose the subject to the disorder inquestion. In the case of an anti-TNFα antibody, or antigen bindingfragment thereof, such as adalimumab, a therapeutically effective amountof the composition comprising a protein with a modulated glycosylationprofile may be administered to treat a disorder in which TNFα activityis detrimental.

A disorder in which TNFα activity is detrimental includes a disorder inwhich inhibition of TNFα activity is expected to alleviate the symptomsand/or progression of the disorder. Such disorders may be evidenced, forexample, by an increase in the concentration of TNFα in a biologicalfluid of a subject suffering from the disorder (e.g., an increase in theconcentration of TNFα in serum, plasma, synovial fluid, etc. of thesubject), which can be detected, for example, using an anti-TNFαantibody.

TNFα has been implicated in the pathophysiology of a wide variety of aTNFα-related disorders including sepsis, infections, autoimmunediseases, transplant rejection and graft-versus-host disease (see e.g.,Moeller, A., et al. (1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024to Moeller et al.; European Patent Publication No. 260 610 B1 byMoeller, A., et al. Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452;Tracey, K. J. and Cerami, A. (1994) Annu. Rev. Med. 45:491-503).Accordingly, the protein with a modulated glycosylation profile of theinvention may be used to treat an autoimmune disease, such as rheumatoidarthritis, juvenile idiopathic arthritis, or psoriatic arthritis, anintestinal disorder, such as Crohn's disease or ulcerative colitis, aspondyloarthropathy, such as ankylosing spondylitis, or a skin disorder,such as psoriasis.

Disorders in which TNFα activity is detrimental are well known in theart and described in detail in U.S. Pat. No. 8,231,876 and U.S. Pat. No.6,090,382, the entire contents of each of which are expresslyincorporated herein by reference. In one embodiment, “a disorder inwhich TNFα activity is detrimental” includes sepsis (including septicshock, endotoxic shock, gram negative sepsis and toxic shock syndrome),autoimmune diseases (including rheumatoid arthritis, rheumatoidspondylitis, osteoarthritis and gouty arthritis, allergy, multiplesclerosis, autoimmune diabetes, autoimmune uveitis, nephrotic syndrome,multisystem autoimmune diseases, lupus (including systemic lupus, lupusnephritis and lupus cerebritis), Crohn's disease and autoimmune hearingloss), infectious diseases (including malaria, meningitis, acquiredimmune deficiency syndrome (AIDS), influenza and cachexia secondary toinfection), allograft rejection and graft versus host disease,malignancy, pulmonary disorders (including adult respiratory distresssyndrome (ARDS), shock lung, chronic pulmonary inflammatory disease,pulmonary sarcoidosis, pulmonary fibrosis, silicosis, idiopathicinterstitial lung disease and chronic obstructive airway disorders(COPD), such as asthma), intestinal disorders (including inflammatorybowel disorders, idiopathic inflammatory bowel disease, Crohn's diseaseand Crohn's disease-related disorders (including fistulas in thebladder, vagina, and skin; bowel obstructions; abscesses; nutritionaldeficiencies; complications from corticosteroid use; inflammation of thejoints; erythem nodosum; pyoderma gangrenosum; lesions of the eye,Crohn's related arthralgias, fistulizing Crohn's indeterminant colitisand pouchitis), cardiac disorders (including ischemia of the heart,heart insufficiency, restenosis, congestive heart failure, coronaryartery disease, angina pectoris, myocardial infarction, cardiovasculartissue damage caused by cardiac arrest, cardiovascular tissue damagecaused by cardiac bypass, cardiogenic shock, and hypertension,atherosclerosis, cardiomyopathy, coronary artery spasm, coronary arterydisease, valvular disease, arrhythmias, and cardiomyopathies),spondyloarthropathies (including ankylosing spondylitis, psoriaticarthritis/spondylitis, enteropathic arthritis, reactive arthritis orReiter's syndrome, and undifferentiated spondyloarthropathies),metabolic disorders (including obesity and diabetes, including type 1diabetes mellitus, type 2 diabetes mellitus, diabetic neuropathy,peripheral neuropathy, diabetic retinopathy, diabetic ulcerations,retinopathy ulcerations and diabetic macrovasculopathy), anemia, pain(including acute and chronic pains, such as neuropathic pain andpost-operative pain, chronic lower back pain, cluster headaches, herpesneuralgia, phantom limb pain, central pain, dental pain,opioid-resistant pain, visceral pain, surgical pain, bone injury pain,pain during labor and delivery, pain resulting from burns, includingsunburn, post partum pain, migraine, angina pain, and genitourinarytract-related pain including cystitis), hepatic disorders (includinghepatitis, alcoholic hepatitis, viral hepatitis, alcoholic cirrhosis, a1antitypsin deficiency, autoimmune cirrhosis, cryptogenic cirrhosis,fulminant hepatitis, hepatitis B and C, and steatohepatitis, cysticfibrosis, primary biliary cirrhosis, sclerosing cholangitis and biliaryobstruction), skin and nail disorders (including psoriasis (includingchronic plaque psoriasis, guttate psoriasis, inverse psoriasis, pustularpsoriasis and other psoriasis disorders), pemphigus vulgaris,scleroderma, atopic dermatitis (eczema), sarcoidosis, erythema nodosum,hidradenitis suppurative, lichen planus, Sweet's syndrome, sclerodermaand vitiligo), vasculitides (including Behcet's disease), and otherdisorders, such as juvenile rheumatoid arthritis (JRA), endometriosis,prostatitis, choroidal neovascularization, sciatica, Sjogren's syndrome,uveitis, wet macular degeneration, osteoporosis, osteoarthritis, activeaxial spondyloarthritis and non-radiographic axial spondyloarthritis.

As used herein, the term “subject” is intended to include livingorganisms, e.g., prokaryotes and eukaryotes. Examples of subjectsinclude mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats,cats, mice, rabbits, rats, and transgenic non-human animals. In specificembodiments of the invention, the subject is a human.

As used herein, the term “treatment” or “treat” refers to boththerapeutic treatment and prophylactic or preventative measures. Thosein need of treatment include those already with the disorder, as well asthose in which the disorder is to be prevented.

In one embodiment, the invention provides a method of administering acomposition comprising a protein with a modulated glycosylation profile,such as an anti-TNFα antibody, or antigen binding fragment thereof, to asubject such that TNFα activity is inhibited or a disorder in which TNFαactivity is detrimental is treated. In one embodiment, the TNFα is humanTNFα and the subject is a human subject. In one embodiment, theanti-TNFα antibody is adalimumab, also referred to as HUMIRA®.

The compositions comprising a protein with a modulated glycosylationprofile can be administered by a variety of methods known in the art.Exemplary routes/modes of administration include subcutaneous injection,intravenous injection or infusion. In certain aspects, a compositioncomprising a protein with a modulated glycosylation profile may beorally administered. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. In certainembodiments it is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit comprising a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic or prophylactic effect to be achieved, and(b) the limitations inherent in the art of compounding such an activecompound for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a composition comprising a proteinwith a modulated glycosylation profile of the invention is 0.01-20mg/kg, or 1-10 mg/kg, or 0.3-1 mg/kg. With respect to a compositioncomprising a protein such as an anti-TNFα antibody with a modulatedglycosylation profile, or antigen-binding portion thereof, such asadalimumab, an exemplary dose is 40 mg every other week. In someembodiments, in particular for treatment of ulcerative colitis orCrohn's disease, an exemplary dose includes an initial dose (Day 1) of160 mg (e.g., four 40 mg injections in one day or two 40 mg injectionsper day for two consecutive days), a second dose two weeks later of 80mg, and a maintenance dose of 40 mg every other week beginning two weekslater. Alternatively, for psoriasis for example, a dosage can include an80 mg initial dose followed by 40 mg every other week starting one weekafter the initial dose.

It is to be noted that dosage values may vary with the type and severityof the condition to be alleviated. It is to be further understood thatfor any particular subject, specific dosage regimens should be adjustedover time according to the individual need and the professional judgmentof the person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition.

IV. Pharmaceutical Formulations Containing Compositions ComprisingProteins with Modulated Glycosylation Profiles of the Invention

The present invention further provides preparations and formulationscomprising compositions comprising a protein with a modulatedglycosylation profile, for example a protein such as an antibody,antigen-binding portion thereof, or a DVD-Ig, with a decreasedfucosylation level or amount and/or an increased mannosylation level oramount. It should be understood that any of the compositions comprisingthe proteins with modulated glycosylation profiles, such as antibodies,antibody fragments and DVD-Igs described herein, may be formulated orprepared as described below. In one embodiment, the antibody is ananti-TNFα antibody, or antigen-binding portion thereof.

In certain embodiments, the compositions comprising a protein with amodulated glycosylation profile, of the invention may be formulated witha pharmaceutically acceptable carrier as pharmaceutical (therapeutic)compositions, and may be administered by a variety of methods known inthe art. As will be appreciated by the skilled artisan, the route and/ormode of administration will vary depending upon the desired results. Theterm “pharmaceutically acceptable carrier” means one or more non-toxicmaterials that do not interfere with the effectiveness of the biologicalactivity of the active ingredients. Such preparations may routinelycontain salts, buffering agents, preservatives, compatible carriers, andoptionally other therapeutic agents. Such pharmaceutically acceptablepreparations may also routinely contain compatible solid or liquidfillers, diluents or encapsulating substances which are suitable foradministration into a human. The term “carrier” denotes an organic orinorganic ingredient, natural or synthetic, with which the activeingredient is combined to facilitate the application. The components ofthe pharmaceutical compositions also are capable of being co-mingledwith the protein with a modulated glycosylation profile (e.g.,antibodies or DVD-Igs) of the present invention, and with each other, ina manner such that there is no interaction which would substantiallyimpair the desired pharmaceutical efficacy.

The compositions comprising a protein with a modulated glycosylationprofile, of the invention are present in a form known in the art andacceptable for therapeutic uses. In one embodiment, a formulation of thecompositions comprising a protein with a modulated glycosylationprofile, of the invention is a liquid formulation. In anotherembodiment, a formulation of the compositions comprising a protein witha modulated glycosylation profile, of the invention is a lyophilizedformulation. In a further embodiment, a formulation of the compositionscomprising a protein with a modulated glycosylation profile, of theinvention is a reconstituted liquid formulation. In one embodiment, aformulation of the compositions comprising a protein with a modulatedglycosylation profile, of the invention is a stable liquid formulation.In one embodiment, a liquid formulation of the compositions comprising aprotein with a modulated glycosylation profile, of the invention is anaqueous formulation. In another embodiment, the liquid formulation isnon-aqueous. In a specific embodiment, a liquid formulation of thecompositions comprising a protein with a modulated glycosylationprofile, of the invention is an aqueous formulation wherein the aqueouscarrier is distilled water.

The formulations of the compositions comprising a protein with amodulated glycosylation profile (e.g., an antibody or a DVD-Ig) in aconcentration resulting in a w/v appropriate for a desired dose. Theprotein with a modulated glycosylation profile may be present in theformulation at a concentration of about 1 mg/ml to about 500 mg/ml,e.g., at a concentration of at least 1 mg/ml, at least 5 mg/ml, at least10 mg/ml, at least 15 mg/ml, at least 20 mg/ml, at least 25 mg/ml, atleast 30 mg/ml, at least 35 mg/ml, at least 40 mg/ml, at least 45 mg/ml,at least 50 mg/ml, at least 55 mg/ml, at least 60 mg/ml, at least 65mg/ml, at least 70 mg/ml, at least 75 mg/ml, at least 80 mg/ml, at least85 mg/ml, at least 90 mg/ml, at least 95 mg/ml, at least 100 mg/ml, atleast 105 mg/ml, at least 110 mg/ml, at least 115 mg/ml, at least 120mg/ml, at least 125 mg/ml, at least 130 mg/ml, at least 135 mg/ml, atleast 140 mg/ml, at least 150 mg/ml, at least 200 mg/ml, at least 250mg/ml, or at least 300 mg/ml.

In a specific embodiment, a formulation of compositions comprising aprotein with a modulated glycosylation profile, of the inventioncomprises at least about 100 mg/ml, at least about 125 mg/ml, at least130 mg/ml, or at least about 150 mg/ml of protein with a modulatedglycosylation profile (e.g., an antibody or DVD-Ig) of the invention.

In one embodiment, the concentration of a protein with a modulatedglycosylation profile (e.g., antibody or DVD-Ig), which is included inthe formulation of the invention, is between about 1 mg/ml and about 25mg/ml, between about 1 mg/ml and about 200 mg/ml, between about 25 mg/mland about 200 mg/ml, between about 50 mg/ml and about 200 mg/ml, betweenabout 75 mg/ml and about 200 mg/ml, between about 100 mg/ml and about200 mg/ml, between about 125 mg/ml and about 200 mg/ml, between about150 mg/ml and about 200 mg/ml, between about 25 mg/ml and about 150mg/ml, between about 50 mg/ml and about 150 mg/ml, between about 75mg/ml and about 150 mg/ml, between about 100 mg/ml and about 150 mg/ml,between about 125 mg/ml and about 150 mg/ml, between about 25 mg/ml andabout 125 mg/ml, between about 50 mg/ml and about 125 mg/ml, betweenabout 75 mg/ml and about 125 mg/ml, between about 100 mg/ml and about125 mg/ml, between about 25 mg/ml and about 100 mg/ml, between about 50mg/ml and about 100 mg/ml, between about 75 mg/ml and about 100 mg/ml,between about 25 mg/ml and about 75 mg/ml, between about 50 mg/ml andabout 75 mg/ml, or between about 25 mg/ml and about 50 mg/ml.

In a specific embodiment, a formulation of the compositions comprising aprotein with a modulated glycosylation profile of the inventioncomprises between about 90 mg/ml and about 110 mg/ml or between about100 mg/ml and about 210 mg/ml of a protein with a modulatedglycosylation profile (e.g., an antibody or DVD-Ig).

The formulations of the compositions comprising a protein with amodulated glycosylation profile of the invention comprising a protein(e.g., an antibody or DVD-Ig) may further comprise one or more activecompounds as necessary for the particular indication being treated,typically those with complementary activities that do not adverselyaffect each other. Such additional active compounds are suitably presentin combination in amounts that are effective for the purpose intended.

The formulations of the compositions comprising a protein with amodulated glycosylation profile may be prepared for storage by mixingthe protein (e.g., antibody or DVD-Ig) having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers, including, but not limited to buffering agents,saccharides, salts, surfactants, solubilizers, polyols, diluents,binders, stabilizers, salts, lipophilic solvents, amino acids,chelators, preservatives, or the like (Goodman and Gilman's ThePharmacological Basis of Therapeutics, 12^(th) edition, L. Brunton, etal. and Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed.(1999)), in the form of lyophilized formulations or aqueous solutions ata desired final concentration. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as histidine, phosphate, citrate,glycine, acetate and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrolidone; amino acids such as glycine, glutamine, asparagine,histidine, arginine, or lysine; monosaccharides, disaccharides, andother carbohydrates including trehalose, glucose, mannose, or dextrins;chelating agents such as EDTA; sugars such as sucrose, mannitol,trehalose or sorbitol; salt-forming counter-ions such as sodium; metalcomplexes (e.g., Zn-protein complexes); and/or non-ionic surfactantssuch as TWEEN, polysorbate 80, PLURONICS™ or polyethylene glycol (PEG).

The buffering agent may be histidine, citrate, phosphate, glycine, oracetate. The saccharide excipient may be trehalose, sucrose, mannitol,maltose or raffinose. The surfactant may be polysorbate 20, polysorbate40, polysorbate 80, or Pluronic F68. The salt may be NaCl, KCl, MgCl₂,or CaCl₂

The formulations of the compositions comprising a protein with amodulated glycosylation profile of the invention may include a bufferingor pH adjusting agent to provide improved pH control. A formulation ofthe invention may have a pH of between about 3.0 and about 9.0, betweenabout 4.0 and about 8.0, between about 5.0 and about 8.0, between about5.0 and about 7.0, between about 5.0 and about 6.5, between about 5.5and about 8.0, between about 5.5 and about 7.0, or between about 5.5 andabout 6.5. In a further embodiment, a formulation of the invention has apH of about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, about 5.1,about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4,about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about7.5, about 8.0, about 8.5, or about 9.0. In a specific embodiment, aformulation of the invention has a pH of about 6.0. One of skill in theart understands that the pH of a formulation generally should not beequal to the isoelectric point of the particular a protein (e.g.,antibody or DVD-Ig) to be used in the formulation.

Typically, the buffering agent is a salt prepared from an organic orinorganic acid or base. Representative buffering agents include, but arenot limited to, organic acid salts such as salts of citric acid,ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinicacid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride,or phosphate buffers. In addition, amino acid components can alsofunction in a buffering capacity. Representative amino acid componentswhich may be utilized in the formulations of the invention as bufferingagents include, but are not limited to, glycine and histidine. Incertain embodiments, the buffering agent is chosen from histidine,citrate, phosphate, glycine, and acetate. In a specific embodiment, thebuffering agent is histidine. In another specific embodiment, thebuffering agent is citrate. In yet another specific embodiment, thebuffering agent is glycine. The purity of the buffering agent should beat least 98%, or at least 99%, or at least 99.5%. As used herein, theterm “purity” in the context of histidine and glycine refers to chemicalpurity of histidine or glycine as understood in the art, e.g., asdescribed in The Merck Index, 13^(th) ed., O'Neil et al. ed. (Merck &Co., 2001).

Buffering agents are typically used at concentrations between about 1 mMand about 200 mM or any range or value therein, depending on the desiredionic strength and the buffering capacity required. The usualconcentrations of conventional buffering agents employed in parenteralformulations can be found in: Pharmaceutical Dosage Form: ParenteralMedications, Volume 1, 2^(nd) Edition, Chapter 5, p. 194, De Luca andBoylan, “Formulation of Small Volume Parenterals”, Table 5: Commonlyused additives in Parenteral Products. In one embodiment, the bufferingagent is at a concentration of about 1 mM, or of about 5 mM, or of about10 mM, or of about 15 mM, or of about 20 mM, or of about 25 mM, or ofabout 30 mM, or of about 35 mM, or of about 40 mM, or of about 45 mM, orof about 50 mM, or of about 60 mM, or of about 70 mM, or of about 80 mM,or of about 90 mM, or of about 100 mM. In one embodiment, the bufferingagent is at a concentration of 1 mM, or of 5 mM, or of 10 mM, or of 15mM, or of 20 mM, or of 25 mM, or of 30 mM, or of 35 mM, or of 40 mM, orof 45 mM, or of 50 mM, or of 60 mM, or of 70 mM, or of 80 mM, or of 90mM, or of 100 mM. In a specific embodiment, the buffering agent is at aconcentration of between about 5 mM and about 50 mM. In another specificembodiment, the buffering agent is at a concentration of between 5 mMand 20 mM.

In certain embodiments, the formulation of the compositions comprising aprotein with a modulated glycosylation profile of the inventioncomprises histidine as a buffering agent. In one embodiment thehistidine is present in the formulation of the invention at aconcentration of at least about 1 mM, at least about 5 mM, at leastabout 10 mM, at least about 20 mM, at least about 30 mM, at least about40 mM, at least about 50 mM, at least about 75 mM, at least about 100mM, at least about 150 mM, or at least about 200 mM histidine. Inanother embodiment, a formulation of the invention comprises betweenabout 1 mM and about 200 mM, between about 1 mM and about 150 mM,between about 1 mM and about 100 mM, between about 1 mM and about 75 mM,between about 10 mM and about 200 mM, between about 10 mM and about 150mM, between about 10 mM and about 100 mM, between about 10 mM and about75 mM, between about 10 mM and about 50 mM, between about 10 mM andabout 40 mM, between about 10 mM and about 30 mM, between about 20 mMand about 75 mM, between about 20 mM and about 50 mM, between about 20mM and about 40 mM, or between about 20 mM and about 30 mM histidine. Ina further embodiment, the formulation comprises about 1 mM, about 5 mM,about 10 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM,about 90 mM, about 100 mM, about 150 mM, or about 200 mM histidine. In aspecific embodiment, a formulation may comprise about 10 mM, about 25mM, or no histidine.

The formulations of the compositions comprising a protein with amodulated glycosylation profile of the invention may comprise acarbohydrate excipient. Carbohydrate excipients can act, e.g., asviscosity enhancing agents, stabilizers, bulking agents, solubilizingagents, and/or the like. Carbohydrate excipients are generally presentat between about 1% to about 99% by weight or volume, e.g., betweenabout 0.1% to about 20%, between about 0.1% to about 15%, between about0.1% to about 5%, between about 1% to about 20%, between about 5% toabout 15%, between about 8% to about 10%, between about 10% and about15%, between about 15% and about 20%, between 0.1% to 20%, between 5% to15%, between 8% to 10%, between 10% and 15%, between 15% and 20%,between about 0.1% to about 5%, between about 5% to about 10%, orbetween about 15% to about 20%. In still other specific embodiments, thecarbohydrate excipient is present at 1%, or at 1.5%, or at 2%, or at2.5%, or at 3%, or at 4%, or at 5%, or at 10%, or at 15%, or at 20%.

Carbohydrate excipients suitable for use in the formulations of theinvention include, but are not limited to, monosaccharides such asfructose, maltose, galactose, glucose, D-mannose, sorbose, and the like;disaccharides, such as lactose, sucrose, trehalose, cellobiose, and thelike; polysaccharides, such as raffinose, melezitose, maltodextrins,dextrans, starches, and the like; and alditols, such as mannitol,xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and the like.In one embodiment, the carbohydrate excipients for use in the presentinvention are chosen from, sucrose, trehalose, lactose, mannitol, andraffinose. In a specific embodiment, the carbohydrate excipient istrehalose. In another specific embodiment, the carbohydrate excipient ismannitol. In yet another specific embodiment, the carbohydrate excipientis sucrose. In still another specific embodiment, the carbohydrateexcipient is raffinose. The purity of the carbohydrate excipient shouldbe at least 98%, or at least 99%, or at least 99.5%.

In a specific embodiment, the formulations of the compositionscomprising a protein with a modulated glycosylation profile of theinvention may comprise trehalose. In one embodiment, a formulation ofthe invention comprises at least about 1%, at least about 2%, at leastabout 4%, at least about 8%, at least about 20%, at least about 30%, orat least about 40% trehalose. In another embodiment, a formulation ofthe invention comprises between about 1% and about 40%, between about 1%and about 30%, between about 1% and about 20%, between about 2% andabout 40%, between about 2% and about 30%, between about 2% and about20%, between about 4% and about 40%, between about 4% and about 30%, orbetween about 4% and about 20% trehalose. In a further embodiment, aformulation of the invention comprises about 1%, about 2%, about 4%,about 6%, about 8%, about 15%, about 20%, about 30%, or about 40%trehalose. In a specific embodiment, a formulation of the inventioncomprises about 4%, about 6% or about 15% trehalose.

In certain embodiments, a formulation of the compositions comprising aprotein with a modulated glycosylation profile of the inventioncomprises an excipient. In a specific embodiment, a formulation of theinvention comprises at least one excipient chosen from: sugar, salt,surfactant, amino acid, polyol, chelating agent, emulsifier andpreservative. In one embodiment, a formulation of the inventioncomprises a salt, e.g., a salt selected from: NaCl, KCl, CaCl₂, andMgCl₂. In a specific embodiment, the formulation comprises NaCl.

A formulation of the compositions comprising a protein with a modulatedglycosylation profile of the invention may comprise at least about 10mM, at least about 25 mM, at least about 50 mM, at least about 75 mM, atleast about 80 mM, at least about 100 mM, at least about 125 mM, atleast about 150 mM, at least about 175 mM, at least about 200 mM, or atleast about 300 mM sodium chloride (NaCl). In a further embodiment, theformulation may comprise between about 10 mM and about 300 mM, betweenabout 10 mM and about 200 mM, between about 10 mM and about 175 mM,between about 10 mM and about 150 mM, between about 25 mM and about 300mM, between about 25 mM and about 200 mM, between about 25 mM and about175 mM, between about 25 mM and about 150 mM, between about 50 mM andabout 300 mM, between about 50 mM and about 200 mM, between about 50 mMand about 175 mM, between about 50 mM and about 150 mM, between about 75mM and about 300 mM, between about 75 mM and about 200 mM, between about75 mM and about 175 mM, between about 75 mM and about 150 mM, betweenabout 100 mM and about 300 mM, between about 100 mM and about 200 mM,between about 100 mM and about 175 mM, or between about 100 mM and about150 mM sodium chloride. In a further embodiment, the formulation maycomprise about 10 mM, about 25 mM, about 50 mM, about 75 mM, about 80mM, about 100 mM, about 125 mM, about 150 mM, about 175 mM, about 200mM, or about 300 mM sodium chloride.

A formulation of the compositions comprising a protein with a modulatedglycosylation profile of the invention may also comprise an amino acid,e.g., lysine, arginine, glycine, histidine or an amino acid salt. Theformulation may comprise at least about 1 mM, at least about 10 mM, atleast about 25 mM, at least about 50 mM, at least about 100 mM, at leastabout 150 mM, at least about 200 mM, at least about 250 mM, at leastabout 300 mM, at least about 350 mM, or at least about 400 mM of anamino acid. In another embodiment, the formulation may comprise betweenabout 1 mM and about 100 mM, between about 10 mM and about 150 mM,between about 25 mM and about 250 mM, between about 25 mM and about 300mM, between about 25 mM and about 350 mM, between about 25 mM and about400 mM, between about 50 mM and about 250 mM, between about 50 mM andabout 300 mM, between about 50 mM and about 350 mM, between about 50 mMand about 400 mM, between about 100 mM and about 250 mM, between about100 mM and about 300 mM, between about 100 mM and about 400 mM, betweenabout 150 mM and about 250 mM, between about 150 mM and about 300 mM, orbetween about 150 mM and about 400 mM of an amino acid. In a furtherembodiment, a formulation of the invention comprises about 1 mM, 1.6 mM,25 mM, about 50 mM, about 100 mM, about 150 mM, about 200 mM, about 250mM, about 300 mM, about 350 mM, or about 400 mM of an amino acid.

The formulations of the compositions comprising a protein with amodulated glycosylation profile of the invention may further comprise asurfactant. The term “surfactant” as used herein refers to organicsubstances having amphipathic structures; namely, they are composed ofgroups of opposing solubility tendencies, typically an oil-solublehydrocarbon chain and a water-soluble ionic group. Surfactants can beclassified, depending on the charge of the surface-active moiety, intoanionic, cationic, and nonionic surfactants. Surfactants are often usedas wetting, emulsifying, solubilizing, and dispersing agents for variouspharmaceutical compositions and preparations of biological materials.Pharmaceutically acceptable surfactants like polysorbates (e.g.,polysorbates 20 or 80); polyoxamers (e.g., poloxamer 188); Triton;sodium octyl glycoside; lauryl-, myristyl-, linoleyl-, orstearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine(e.g., lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUA™ series (Mona Industries, Inc.,Paterson, N.J.), polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g., PLURONICS™ PF68, etc.), canoptionally be added to the formulations of the invention to reduceaggregation. In one embodiment, a formulation of the invention comprisesPolysorbate 20, Polysorbate 40, Polysorbate 60, or Polysorbate 80.Surfactants are particularly useful if a pump or plastic container isused to administer the formulation. The presence of a pharmaceuticallyacceptable surfactant mitigates the propensity for the protein toaggregate. The formulations may comprise a polysorbate which is at aconcentration ranging from between about 0.001% to about 1%, or about0.001% to about 0.1%, or about 0.01% to about 0.1%. In other specificembodiments, the formulations of the invention comprise a polysorbatewhich is at a concentration of 0.001%, or 0.002%, or 0.003%, or 0.004%,or 0.005%, or 0.006%, or 0.007%, or 0.008%, or 0.009%, or 0.01%, or0.015%, or 0.02%.

The formulations of the compositions comprising a protein with amodulated glycosylation profile of the invention may optionally furthercomprise other common excipients and/or additives including, but notlimited to, diluents, binders, stabilizers, lipophilic solvents,preservatives, adjuvants, or the like. Pharmaceutically acceptableexcipients and/or additives may be used in the formulations of theinvention. Commonly used excipients/additives, such as pharmaceuticallyacceptable chelators (for example, but not limited to, EDTA, DTPA orEGTA) can optionally be added to the formulations of the invention toreduce aggregation. These additives are particularly useful if a pump orplastic container is used to administer the formulation.

Preservatives, such as phenol, m-cresol, p-cresol, o-cresol,chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,formaldehyde, chlorobutanol, magnesium chloride (for example, but notlimited to, hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl andthe like), benzalkonium chloride, benzethonium chloride, sodiumdehydroacetate and thimerosal, or mixtures thereof can optionally beadded to the formulations of the invention at any suitable concentrationsuch as between about 0.001% to about 5%, or any range or value therein.The concentration of preservative used in the formulations of theinvention is a concentration sufficient to yield a microbial effect.Such concentrations are dependent on the preservative selected and arereadily determined by the skilled artisan.

Other contemplated excipients/additives, which may be utilized in theformulations of the invention include, for example, flavoring agents,antimicrobial agents, sweeteners, antioxidants, antistatic agents,lipids such as phospholipids or fatty acids, steroids such ascholesterol, protein excipients such as serum albumin (human serumalbumin (HSA), recombinant human albumin (rHA), gelatin, casein,salt-forming counterions such as sodium and the like. These andadditional known pharmaceutical excipients and/or additives suitable foruse in the formulations of the invention are known in the art, e.g., aslisted in “Remington: The Science & Practice of Pharmacy”, 21^(st) ed.,Lippincott Williams & Wilkins, (2005), and in the “Physician's DeskReference”, 60^(th) ed., Medical Economics, Montvale, N.J. (2005).Pharmaceutically acceptable carriers can be routinely selected that aresuitable for the mode of administration, solubility and/or stability ofprotein with a modulated glycosylation profile (e.g., an antibody orDVD-Ig), as well known those in the art or as described herein.

In one embodiment, the compositions comprising a protein with amodulated glycosylation profile of the invention are formulated with thesame or similar excipients and buffers as are present in the commercialadalimumab (HUMIRA®) formulation, as described in the “Highlights ofPrescribing Information” for HUMIRA® (adalimumab) Injection (RevisedJanuary 2008) the contents of which are hereby incorporated herein byreference. For example, each prefilled syringe of HUMIRA®, which isadministered subcutaneously, delivers 0.8 mL (40 mg) of drug product tothe subject. Each 0.8 mL of HUMIRA® contains 40 mg adalimumab, 4.93 mgsodium chloride, 0.69 mg monobasic sodium phosphate dihydrate, 1.22 mgdibasic sodium phosphate dihydrate, 0.24 mg sodium citrate, 1.04 mgcitric acid monohydrate, 9.6 mg mannitol, 0.8 mg polysorbate 80, andwater for Injection, USP. Sodium hydroxide is added as necessary toadjust pH.

It will be understood by one skilled in the art that the formulations ofthe compositions comprising a protein with a modulated glycosylationprofile of the invention may be isotonic with human blood, wherein theformulations of the invention have essentially the same osmotic pressureas human blood. Such isotonic formulations will generally have anosmotic pressure from about 250 mOSm to about 350 mOSm. Isotonicity canbe measured by, for example, using a vapor pressure or ice-freezing typeosmometer. Tonicity of a formulation is adjusted by the use of tonicitymodifiers. “Tonicity modifiers” are those pharmaceutically acceptableinert substances that can be added to the formulation to provide anisotonity of the formulation. Tonicity modifiers suitable for thisinvention include, but are not limited to, saccharides, salts and aminoacids.

In certain embodiments, the formulations of the compositions comprisinga protein with a modulated glycosylation profile of the invention havean osmotic pressure from about 100 mOSm to about 1200 mOSm, or fromabout 200 mOSm to about 1000 mOSm, or from about 200 mOSm to about 800mOSm, or from about 200 mOSm to about 600 mOSm, or from about 250 mOSmto about 500 mOSm, or from about 250 mOSm to about 400 mOSm, or fromabout 250 mOSm to about 350 mOSm.

The concentration of any one component or any combination of variouscomponents, of the formulations of the compositions comprising a proteinwith a modulated glycosylation profile of the invention is adjusted toachieve the desired tonicity of the final formulation. For example, theratio of the carbohydrate excipient to protein with a modulatedglycosylation profile (e.g., antibody or DVD-Ig) may be adjustedaccording to methods known in the art (e.g., U.S. Pat. No. 6,685,940).In certain embodiments, the molar ratio of the carbohydrate excipient toprotein with a modulated glycosylation profile (e.g., antibody orDVD-Ig) may be from about 100 moles to about 1000 moles of carbohydrateexcipient to about 1 mole of protein with a modulated glycosylationprofile, or from about 200 moles to about 6000 moles of carbohydrateexcipient to about 1 mole of protein with a modulated glycosylationprofile, or from about 100 moles to about 510 moles of carbohydrateexcipient to about 1 mole of protein with a modulated glycosylationprofile, or from about 100 moles to about 600 moles of carbohydrateexcipient to about 1 mole of protein with a modulated glycosylationprofile.

The desired isotonicity of the final formulation may also be achieved byadjusting the salt concentration of the formulations. Pharmaceuticallyacceptable salts and those suitable for this invention as tonicitymodifiers include, but are not limited to, sodium chloride, sodiumsuccinate, sodium sulfate, potassium chloride, magnesium chloride,magnesium sulfate, and calcium chloride. In specific embodiments,formulations of the invention comprise NaCl, MgCl₂, and/or CaCl₂. In oneembodiment, concentration of NaCl is between about 75 mM and about 150mM. In another embodiment, concentration of MgCl₂ is between about 1 mMand about 100 mM. Pharmaceutically acceptable amino acids includingthose suitable for this invention as tonicity modifiers include, but arenot limited to, proline, alanine, L-arginine, asparagine, L-asparticacid, glycine, serine, lysine, and histidine.

In one embodiment the formulations of the compositions comprising aprotein with a modulated glycosylation profile of the invention arepyrogen-free formulations which are substantially free of endotoxinsand/or related pyrogenic substances. Endotoxins include toxins that areconfined inside a microorganism and are released only when themicroorganisms are broken down or die. Pyrogenic substances also includefever-inducing, thermostable substances (glycoproteins) from the outermembrane of bacteria and other microorganisms. Both of these substancescan cause fever, hypotension and shock if administered to humans. Due tothe potential harmful effects, even low amounts of endotoxins must beremoved from intravenously administered pharmaceutical drug solutions.The Food & Drug Administration (“FDA”) has set an upper limit of 5endotoxin units (EU) per dose per kilogram body weight in a single onehour period for intravenous drug applications (The United StatesPharmacopeial Convention, Pharmacopeial Forum 26 (1):223 (2000)). Whentherapeutic proteins are administered in amounts of several hundred orthousand milligrams per kilogram body weight, as can be the case withproteins of interest (e.g., antibodies), even trace amounts of harmfuland dangerous endotoxin must be removed. In certain specificembodiments, the endotoxin and pyrogen levels in the composition areless than 10 EU/mg, or less than 5 EU/mg, or less than 1 EU/mg, or lessthan 0.1 EU/mg, or less than 0.01 EU/mg, or less than 0.001 EU/mg.

When used for in vivo administration, the formulations of thecompositions comprising a protein with a modulated glycosylation profileof the invention should be sterile. The formulations of the inventionmay be sterilized by various sterilization methods, including sterilefiltration, radiation, etc. In one embodiment, the protein with amodulated glycosylation profile (e.g., antibody or DVD-Ig) formulationis filter-sterilized with a presterilized 0.22-micron filter. Sterilecompositions for injection can be formulated according to conventionalpharmaceutical practice as described in “Remington: The Science &Practice of Pharmacy”, 21^(st) ed., Lippincott Williams & Wilkins,(2005). Formulations comprising proteins of interest (e.g., antibody orDVD-Ig.), such as those disclosed herein, ordinarily will be stored inlyophilized form or in solution. It is contemplated that sterilecompositions comprising proteins of interest (e.g., antibody or DVD-Ig)are placed into a container having a sterile access port, for example,an intravenous solution bag or vial having an adapter that allowsretrieval of the formulation, such as a stopper pierceable by ahypodermic injection needle. In one embodiment, a composition of theinvention is provided as a pre-filled syringe.

In one embodiment, a formulation of the compositions comprising aprotein with a modulated glycosylation profile of the invention is alyophilized formulation. The term “lyophilized” or “freeze-dried”includes a state of a substance that has been subjected to a dryingprocedure such as lyophilization, where at least 50% of moisture hasbeen removed.

The phrase “bulking agent” includes a compound that is pharmaceuticallyacceptable and that adds bulk to a lyo cake. Bulking agents known to theart include, for example, carbohydrates, including simple sugars such asdextrose, ribose, fructose and the like, alcohol sugars such asmannitol, inositol and sorbitol, disaccharides including trehalose,sucrose and lactose, naturally occurring polymers such as starch,dextrans, chitosan, hyaluronate, proteins (e.g., gelatin and serumalbumin), glycogen, and synthetic monomers and polymers.

A “lyoprotectant” is a molecule which, when combined with a protein witha modulated glycosylation profile (such as an antibody or DVD-Ig of theinvention), significantly prevents or reduces chemical and/or physicalinstability of the protein upon lyophilization and subsequent storage.Lyoprotectants include, but are not limited to, sugars and theircorresponding sugar alcohols; an amino acid such as monosodium glutamateor histidine; a methylamine such as betaine; a lyotropic salt such asmagnesium sulfate; a polyol such as trihydric or higher molecular weightsugar alcohols, e.g., glycerin, dextran, erythritol, glycerol, arabitol,xylitol, sorbitol, and mannitol; propylene glycol; polyethylene glycol;PLURONICS™; and combinations thereof. Additional examples oflyoprotectants include, but are not limited to, glycerin and gelatin,and the sugars mellibiose, melezitose, raffinose, mannotriose andstachyose. Examples of reducing sugars include, but are not limited to,glucose, maltose, lactose, maltulose, iso-maltulose and lactulose.Examples of non-reducing sugars include, but are not limited to,non-reducing glycosides of polyhydroxy compounds selected from sugaralcohols and other straight chain polyalcohols. Examples of sugaralcohols include, but are not limited to, monoglycosides, compoundsobtained by reduction of disaccharides such as lactose, maltose,lactulose and maltulose. The glycosidic side group can be eitherglucosidic or galactosidic. Additional examples of sugar alcoholsinclude, but are not limited to, glucitol, maltitol, lactitol andiso-maltulose. In specific embodiments, trehalose or sucrose is used asa lyoprotectant.

The lyoprotectant is added to the pre-lyophilized formulation in a“lyoprotecting amount” which means that, following lyophilization of theprotein in the presence of the lyoprotecting amount of thelyoprotectant, the protein essentially retains its physical and chemicalstability and integrity upon lyophilization and storage.

In one embodiment, the molar ratio of a lyoprotectant (e.g., trehalose)and protein with a modulated glycosylation profile (e.g., antibody orDVD-Ig) molecules of a formulation of the invention is at least about10, at least about 50, at least about 100, at least about 200, or atleast about 300. In another embodiment, the molar ratio of alyoprotectant (e.g., trehalose) and protein with a modulatedglycosylation profile molecules of a formulation of the invention isabout 1, is about 2, is about 5, is about 10, about 50, about 100, about200, or about 300.

A “reconstituted” formulation is one which has been prepared bydissolving a lyophilized protein with a modulated glycosylation profile(e.g., antibody or DVD-Ig) formulation in a diluent such that theprotein with a modulated glycosylation profile is dispersed in thereconstituted formulation. The reconstituted formulation is suitable foradministration (e.g., parenteral administration) to a patient to betreated with the protein with a modulated glycosylation profile and, incertain embodiments of the invention, may be one which is suitable forintravenous administration.

The “diluent” of interest herein is one which is pharmaceuticallyacceptable (safe and non-toxic for administration to a human) and isuseful for the preparation of a liquid formulation, such as aformulation reconstituted after lyophilization. In some embodiments,diluents include, but are not limited to, sterile water, bacteriostaticwater for injection (BWFI), a pH buffered solution (e.g.,phosphate-buffered saline), sterile saline solution, Ringer's solutionor dextrose solution. In an alternative embodiment, diluents can includeaqueous solutions of salts and/or buffers.

In certain embodiments, a formulation of the compositions comprising aprotein with a modulated glycosylation profile of the invention is alyophilized formulation comprising a protein with a modulatedglycosylation profile (e.g., antibody or DVD-Ig) of the invention,wherein at least about 90%, at least about 95%, at least about 97%, atleast about 98%, or at least about 99% of the protein with a modulatedglycosylation profile may be recovered from a vial upon shaking the vialfor 4 hours at a speed of 400 shakes per minute wherein the vial isfilled to half of its volume with the formulation. In anotherembodiment, a formulation of the invention is a lyophilized formulationcomprising a protein with a modulated glycosylation profile of theinvention, wherein at least about 90%, at least about 95%, at leastabout 97%, at least about 98%, or at least about 99% of the protein witha modulated glycosylation profile may be recovered from a vial uponsubjecting the formulation to three freeze/thaw cycles wherein the vialis filled to half of its volume with the formulation. In a furtherembodiment, a formulation of the invention is a lyophilized formulationcomprising a protein with a modulated glycosylation profile of theinvention, wherein at least about 90%, at least about 95%, at leastabout 97%, at least about 98%, or at least about 99% of the protein witha modulated glycosylation profile may be recovered by reconstituting alyophilized cake generated from the formulation.

In one embodiment, a reconstituted liquid formulation may comprise aprotein with a modulated glycosylation profile (e.g., antibody orDVD-Ig) of the invention at the same concentration as thepre-lyophilized liquid formulation.

In another embodiment, a reconstituted liquid formulation may comprise aprotein with a modulated glycosylation profile (e.g., antibody orDVD-Ig) of the invention at a higher concentration than thepre-lyophilized liquid formulation, e.g., about 2 fold, about 3 fold,about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold,about 9 fold, or about 10 fold higher concentration of a protein with amodulated glycosylation profile than the pre-lyophilized liquidformulation.

In yet another embodiment, a reconstituted liquid formulation maycomprise a protein with a modulated glycosylation profile (e.g.,antibody or DVD-Ig) of the invention at a lower concentration than thepre-lyophilized liquid formulation, e.g., about 2 fold, about 3 fold,about 4 fold, about 5 fold, about 6 fold, about 7 fold, about 8 fold,about 9 fold or about 10 fold lower concentration of a protein with amodulated glycosylation profile than the pre-lyophilized liquidformulation.

The pharmaceutical formulations of the compositions comprising a proteinwith a modulated glycosylation profile, of the invention are typicallystable formulations, e.g., stable at room temperature.

The terms “stability” and “stable” as used herein in the context of aformulation comprising a protein with a modulated glycosylation profile(e.g., an antibody or DVD-Ig) of the invention refer to the resistanceof the protein in the formulation to aggregation, degradation orfragmentation under given manufacture, preparation, transportation andstorage conditions. The “stable” formulations of the invention retainbiological activity under given manufacture, preparation, transportationand storage conditions. The stability of the protein with a modulatedglycosylation profile can be assessed by degrees of aggregation,degradation or fragmentation, as measured by HPSEC, static lightscattering (SLS), Fourier Transform Infrared Spectroscopy (FTIR),circular dichroism (CD), urea unfolding techniques, intrinsic tryptophanfluorescence, differential scanning calorimetry, and/or ANS bindingtechniques, compared to a reference formulation. For example, areference formulation may be a reference standard frozen at −70° C.consisting of 10 mg/ml of a protein with a modulated glycosylationprofile of the invention in PBS.

Therapeutic formulations of the compositions comprising a protein with amodulated glycosylation profile of the invention may be formulated for aparticular dosage. Dosage regimens may be adjusted to provide theoptimum desired response (e.g., a therapeutic response). For example, asingle bolus may be administered, several divided doses may beadministered over time or the dose may be proportionally reduced orincreased as indicated by the exigencies of the therapeutic situation.It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the protein with a modulated glycosylation profile(e.g., antibody or DVD-Ig) of the invention, and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such a protein with a modulated glycosylationprofile for the treatment of sensitivity in individuals.

Therapeutic compositions of the compositions comprising a protein with amodulated glycosylation profile of the invention, can be formulated forparticular routes of administration, such as oral, nasal, pulmonary,topical (including buccal and sublingual), rectal, vaginal and/orparenteral administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods knownin the art of pharmacy. The amount of active ingredient which can becombined with a carrier material to produce a single dosage form willvary depending upon the subject being treated, and the particular modeof administration. The amount of active ingredient which can be combinedwith a carrier material to produce a single dosage form will generallybe that amount of the composition which produces a therapeutic effect.By way of example, in certain embodiments, the proteins with modulatedglycosylation profiles (including fragments of the protein with amodulated glycosylation profile) are formulated for intravenousadministration. In certain other embodiments, the proteins withmodulated glycosylation profiles (e.g., antibody or DVD-Ig), of theinvention, including fragments of the proteins with modulatedglycosylation profiles (e.g., antibody fragments) of the invention, areformulated for local delivery to the cardiovascular system, for example,via catheter, stent, wire, intramyocardial delivery, intrapericardialdelivery, or intraendocardial delivery.

Formulations of the compositions comprising a protein with a modulatedglycosylation profile of the invention, which are suitable for topicalor transdermal administration include powders, sprays, ointments,pastes, creams, lotions, gels, solutions, patches and inhalants. Theactive compound may be mixed under sterile conditions with apharmaceutically acceptable carrier, and with any preservatives,buffers, or propellants which may be required (U.S. Pat. Nos. 7,378,110;7,258,873; 7,135,180; 7,923,029; and US Publication No. 20040042972).

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal, epidural and intrasternal injection andinfusion.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the compositions comprising a protein with a modulatedglycosylation profile of the invention, may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

In certain embodiments, the proteins with modulated glycosylationprofiles (e.g., antibody or DVD-Ig) of the invention can be formulatedto ensure proper distribution in vivo. For example, the blood-brainbarrier (BBB) excludes many highly hydrophilic compounds. To ensure thatthe therapeutic compounds of the invention can cross the BBB (ifdesired), they can be formulated, for example, in liposomes. For methodsof manufacturing liposomes, see, e.g., U.S. Pat. Nos. 4,522,811;5,374,548; 5,399,331. The liposomes may comprise one or more moietieswhich are selectively transported into specific cells or organs, thusenhance targeted drug delivery (see, e.g., V. V. Ranade (1989) J. Clin.Pharmacol. 29:685). Exemplary targeting moieties include folate orbiotin (see, e.g., U.S. Pat. No. 5,416,016); mannosides (Umezawa et al.,(1988) Biochem. Biophys. Res. Commun. 153:1038); antibodies (P. G.Bloeman et al. (1995) FEBS Lett. 357:140; M. Owais et al. (1995)Antimicrob. Agents Chemother. 39:180); surfactant Protein A receptor(Briscoe et al. (1995) Am. J. Physiol. 1233:134), different species ofwhich may comprise the formulations of the invention, as well ascomponents of the invented molecules; p120 (Schreier et al. (1994) J.Biol. Chem. 269:9090); see also K. Keinanen; M. L. Laukkanen (1994) FEBSLett. 346:123; J. J. Killion; I. J. Fidler (1994) Immunomethods 4:273.In one embodiment of the invention, the therapeutic compounds of theinvention are formulated in liposomes; in another embodiment, theliposomes include a targeting moiety. In another embodiment, thetherapeutic compounds in the liposomes are delivered by bolus injectionto a site proximal to the desired area. When administered in thismanner, the composition must be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and may be preserved against the contaminatingaction of microorganisms such as bacteria and fungi. Additionally oralternatively, the proteins with modulated glycosylation profiles (e.g.,antibodies or DVD-Igs) of the invention may be delivered locally to thebrain to mitigate the risk that the blood brain barrier slows effectivedelivery.

In certain embodiments, the compositions comprising a protein with amodulated glycosylation profile of the invention may be administeredwith medical devices known in the art. For example, in certainembodiments a protein with a modulated glycosylation profile (e.g.,antibody or DVD-Ig) or a fragment of protein with a modulatedglycosylation profile (e.g., antibody fragment) is administered locallyvia a catheter, stent, wire, or the like. For example, in oneembodiment, a therapeutic composition of the invention can beadministered with a needleless hypodermic injection device, such as thedevices disclosed in U.S. Pat. Nos. 5,399,163; 5,383,851; 5,312,335;5,064,413; 4,941,880; 4,790,824; 4,596,556. Examples of well-knownimplants and modules useful in the present invention include: U.S. Pat.No. 4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,486,194,which discloses a therapeutic device for administering medicants throughthe skin; U.S. Pat. No. 4,447,233, which discloses a medication infusionpump for delivering medication at a precise infusion rate; U.S. Pat. No.4,447,224, which discloses a variable flow implantable infusionapparatus for continuous drug delivery; U.S. Pat. No. 4,439,196, whichdiscloses an osmotic drug delivery system having multi-chambercompartments; and U.S. Pat. No. 4,475,196, which discloses an osmoticdrug delivery system. Many other such implants, delivery systems, andmodules are known to those skilled in the art.

The efficient dosages and the dosage regimens for the compositionscomprising a protein with a modulated glycosylation profile of theinvention depend on the disease or condition to be treated and can bedetermined by the persons skilled in the art. One of ordinary skill inthe art would be able to determine such amounts based on such factors asthe subject's size, the severity of the subject's symptoms, and theparticular composition or route of administration selected.

VI. Kits and Articles of Manufacture Comprising the CompositionsComprising Proteins with Modulated Glycosylation Profiles of theInvention

Also within the scope of the present invention are kits comprising thecompositions comprising a protein with a modulated glycosylationprofile, for example a protein such as an antibody, antigen-bindingportion thereof, or a DVD-Ig, with a decreased fucosylation level oramount and/or an increased mannosylation level or amount of theinvention and instructions for use. The term “kit” as used herein refersto a packaged product comprising components with which to administer theprotein with a modulated glycosylation profile (e.g., antibody, orantigen-binding portion thereof, or DVD-Ig), of the invention fortreatment of a disease or disorder. The kit may comprise a box orcontainer that holds the components of the kit. The box or container isaffixed with a label or a Food and Drug Administration approvedprotocol. The box or container holds components of the invention whichmay be contained within plastic, polyethylene, polypropylene, ethylene,or propylene vessels. The vessels can be capped-tubes or bottles. Thekit can also include instructions for administering a protein with amodulated glycosylation profile (e.g., an antibody or a DVD-Ig) of theinvention.

The kit can further contain one more additional reagents, such as animmunosuppressive reagent, a cytotoxic agent or a radiotoxic agent orone or more additional proteins of interest of the invention (e.g., anantibody having a complementary activity which binds to an epitope inthe TNFα antigen distinct from a first anti-TNFα antibody). Kitstypically include a label indicating the intended use of the contents ofthe kit. The term label includes any writing, or recorded materialsupplied on or with the kit, or which otherwise accompanies the kit.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with a liquid formulation or lyophilizedformulation of a protein with a modulated glycosylation profile (e.g.,an antibody, or antibody fragment thereof, or a DVD-Ig) of theinvention. In one embodiment, a container filled with a liquidformulation of the invention is a pre-filled syringe. In a specificembodiment, the formulations of the invention are formulated in singledose vials as a sterile liquid. For example, the formulations may besupplied in 3 cc USP Type I borosilicate amber vials (WestPharmaceutical Services—Part No. 6800-0675) with a target volume of 1.2mL. Optionally associated with such container(s) can be a notice in theform prescribed by a governmental agency regulating the manufacture, useor sale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

In one embodiment, a container filled with a liquid formulation of theinvention is a pre-filled syringe. Any pre-filled syringe known to oneof skill in the art may be used in combination with a liquid formulationof the invention. Pre-filled syringes that may be used are described in,for example, but not limited to, PCT Publications WO05032627,WO08094984, WO9945985, WO03077976, U.S. Pat. No. 6,792,743, U.S. Pat.No. 5,607,400, U.S. Pat. No. 5,893,842, U.S. Pat. No. 7,081,107, U.S.Pat. No. 7,041,087, U.S. Pat. No. 5,989,227, U.S. Pat. No. 6,807,797,U.S. Pat. No. 6,142,976, U.S. Pat. No. 5,899,889, U.S. Pat. No.7,699,811, U.S. Pat. No. 7,540,382, U.S. Pat. No. 7,998,120, U.S. Pat.No. 7,645,267, and US Patent Publication No. US20050075611. Pre-filledsyringes may be made of various materials. In one embodiment apre-filled syringe is a glass syringe. In another embodiment apre-filled syringe is a plastic syringe. One of skill in the artunderstands that the nature and/or quality of the materials used formanufacturing the syringe may influence the stability of a proteinformulation stored in the syringe. For example, it is understood thatsilicon based lubricants deposited on the inside surface of the syringechamber may affect particle formation in the protein formulation. In oneembodiment, a pre-filled syringe comprises a silicone based lubricant.In one embodiment, a pre-filled syringe comprises baked on silicone. Inanother embodiment, a pre-filled syringe is free from silicone basedlubricants. One of skill in the art also understands that small amountsof contaminating elements leaching into the formulation from the syringebarrel, syringe tip cap, plunger or stopper may also influence stabilityof the formulation. For example, it is understood that tungstenintroduced during the manufacturing process may adversely affectformulation stability. In one embodiment, a pre-filled syringe maycomprise tungsten at a level above 500 ppb. In another embodiment, apre-filled syringe is a low tungsten syringe. In another embodiment, apre-filled syringe may comprise tungsten at a level between about 500ppb and about 10 ppb, between about 400 ppb and about 10 ppb, betweenabout 300 ppb and about 10 ppb, between about 200 ppb and about 10 ppb,between about 100 ppb and about 10 ppb, between about 50 ppb and about10 ppb, between about 25 ppb and about 10 ppb.

In certain embodiments, kits comprising a protein with a modulatedglycosylation profile such as a decreased fucosylation level or amountand/or an increased mannosylation level or amount (e.g., an antibody orDVD-Ig) of the invention are also provided that are useful for variouspurposes, e.g., research and diagnostic including for purification orimmunoprecipitation of a protein with a modulated glycosylation profilefrom cells, detection of the protein with a modulated glycosylationprofile in vitro or in vivo. For isolation and purification of a proteinwith a modulated glycosylation profile, the kit may contain an antibodycoupled to beads (e.g., sepharose beads). Kits may be provided whichcontain the antibodies for detection and quantitation of a protein witha modulated glycosylation profile in vitro, e.g., in an ELISA or aWestern blot. As with the article of manufacture, the kit comprises acontainer and a label or package insert on or associated with thecontainer. The container holds a composition comprising at least oneprotein with a modulated glycosylation profile (e.g., antibody orDVD-Ig) of the invention. Additional containers may be included thatcontain, e.g., diluents and buffers, control proteins with modulatedglycosylation profiles (e.g., antibody or DVD-Ig). The label or packageinsert may provide a description of the composition as well asinstructions for the intended in vitro or diagnostic use.

The present invention also encompasses a finished packaged and labeledpharmaceutical product. This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial, pre-filled syringe or other container that ishermetically sealed. In one embodiment, the unit dosage form is providedas a sterile particulate free solution comprising a protein with amodulated glycosylation profile (e.g., an antibody or DVD-Ig) that issuitable for parenteral administration. In another embodiment, the unitdosage form is provided as a sterile lyophilized powder comprising aprotein with a modulated glycosylation profile (e.g., an antibody orDVD-Ig) that is suitable for reconstitution.

In one embodiment, the unit dosage form is suitable for intravenous,intramuscular, intranasal, oral, topical or subcutaneous delivery. Thus,the invention encompasses sterile solutions suitable for each deliveryroute. The invention further encompasses sterile lyophilized powdersthat are suitable for reconstitution.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician or patient on how to appropriately prevent or treat thedisease or disorder in question, as well as how and how frequently toadminister the pharmaceutical. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures, and other monitoring information.

Specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, pre-filled syringe, sprayer, insufflator, intravenous (i.v.)bag, envelope and the like; and at least one unit dosage form of apharmaceutical agent contained within the packaging material, whereinthe pharmaceutical agent comprises a liquid formulation containing aprotein with a modulated glycosylation profile (e.g., an antibody orDVD-Ig). The packaging material includes instruction means whichindicate how that the protein with a modulated glycosylation profile(e.g., antibody or DVD-Ig) can be used to prevent, treat and/or manageone or more symptoms associated with a disease or disorder.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting.

EXAMPLES Example 1 1. Materials & Methods

Cell Culture

Two recombinant Chinese Hamster Ovary (CHO) cell lines expressing twodifferent humanized monoclonal antibodies (Antibody 1, Antibody 2) wereevaluated in two different culture vessels (shaker flasks and laboratoryscale bioreactors). Both cell lines were of CHO DUX-B11 origin based ona dhfr (dihydrofolate reductase) expression system. Both cell lines werecultured in the same chemically defined basal media (CDBM), with CellLine 1 also utilizing a chemically-defined feed media (CDFM). Each ofthe respective media were supplemented with selected monosaccharides andoligosaccharides to evaluate their potential impact on the resultingN-glycan oligosaccharide profile. In preparing the cultures, the celllines were serially expanded through separate seed train inoculums togenerate enough cells for inoculation. Process conditions utilizedduring the cultures were slightly different between the two differentcell lines as shown in Table 1, but similar between each cell line andthe respective non-sugar supplemented control conditions.

TABLE 1 Summary of cell culture process conditions & sugarsupplementation details Cell Line 1 Cell Line 2 Culture Vessel 250 mL250 mL 3 L lab-scale 250 mL shaker flask shaker flask bioreactors shakerflask Culture Mode Fedbatch Fedbatch Fedbatch Semi-Batch^(a) InitialCulture 36 36 36 36 Temperature (° C.) Dissolved N/A^(b) N/A^(b) 30N/A^(b) Oxygen (%) pH N/A^(b) N/A^(b) 6.9 N/A^(b) Sugar Sucrose TagatoseSucrose Sucrose Supplements Tagatose Evaluated^(c) Supplement 0, 30, 50,70 0, 30, 50, 70 0, 50 0, 7, 15, 30 Concentrations (mM) ^(a)No feedmedia utilized; concentrated glucose solution addition utilized whenglucose levels dropped below 3 g/L. ^(b)Cultures run in CO₂ incubatorsat 5% CO₂ in air; pH and DO parameters were not controlled, and thus didnot have setpoint values. ^(c)Supplements added to bothchemically-defined basal & feed media (when used).

Viable cell density (VCD) and cell viability values were measuredthrough trypan blue exclusion via Cedex automated cell counters (RocheApplied Science, Indianapolis, Ind.), glucose and lactate values weremeasured with a ABL-805 (Radiometer Medical, Denmark) blood gasanalyzer. Offline pH, dissolved oxygen (DO), and pCO₂ measurements wereperformed with a ABL-805 (Radiometer Medical, Denmark) blood gasanalyzer. Osmolality was measured on a Multi-Osmette 2430 osmometer(Precision Systems, Natick, Mass.).

Protein A Affinity Chromatography

Antibody titers were measured from crude cell culture harvests on aPoros A™ (Life Technologies, Carlsbad, Calif.) affinity column using anAgilent (Santa Clara, Calif.) 1200 Series HPLC, or equivalent, operatingwith a low pH, step elution gradient with detection at 280 nm. Absoluteconcentrations were assigned with respect to reference standardcalibration curves.

Purified antibodies subjected to additional analytical characterizationwere purified using MabSelect™ Protein A (GE Healthcare, Piscataway,N.J.) using a low pH, step elution gradient, followed by buffer exchangeusing Corning Lifesciences (Tewksbury, Mass.) Spin Concentrator X UFcolumns according to the manufacturers' recommended procedures.

N-Glycan Oligosaccharide Profiling

Approximately 200 μg of Protein A purified samples from Cell Lines 1 and2 were treated with N-glycanase at 37° C. overnight to remove theN-glycans from the protein. The protein was precipitated and thesupernatant was taken for subsequent chemical derivatization of thereducing end of the released glycans with 2-aminobenzamide (2-AB) dye.Following the derivatization step, the excess label was removed usingclean up cartridges and the samples were analyzed using normal phaseHPLC with fluorescent detection. Mobile phase A was 100% acetonitrileand mobile phase B was 50 mM ammonium formate pH 4.4. The glycans wereeluted from a polyamide column (Prozyme, Hayward, Calif.) using ashallow gradient. The labeled glycans were detected using a fluorescencedetector with an excitation wavelength of 330 nm and an emissionwavelength of 420 nm.

2. Results

Shake Flask Screening of Select Monosaccharides and Oligosaccharides

Cell Line 1 was cultured in shake flasks in fedbatch mode after anabbreviated seed train. Sucrose and tagatose were supplemented intochemically-defined basal and feed media at concentrations of 30, 50, and70 mM, and compared to an unsupplemented control condition. The viablecell density (VCD), viability, and harvest titer results are shown inFIGS. 3A-3C and FIGS. 5A-5C for the sucrose, and tagatose conditions,respectively. Both sugars facilitated a slight decrease in VCD, with thehigher concentrations also supporting the lowest peak VCD. Viabilityresults were however more comparable to the control, unsupplementedconditions, with the exception of the 70 mM sucrose condition, whichresulted in a decrease in cell viability that was larger, and earlier inthe culture, compared to control conditions. Harvest titer resultsdemonstrated that 30 mM and 50 mM sucrose did not adversely impacttiter, but 70 mM sucrose did reduce the harvest titer significantly. 30mM tagatose did not adversely impact harvest titer, but 50 mM and 70 mMtagatose did. Collectively, these results indicate that at certainconcentrations of sucrose (e.g., 30 mM and 50 mM) and tagatose (e.g., 30mM) there is a decrease in viable cell density, however percent cellviability and harvest titer profile remain comparable to unsupplementedculture conditions. As such, the upper threshold of sucrose or tagatoseconcentration should not be exceeded.

There was a significant impact on the overall N-glycan distribution whencell cultures were supplemented with sucrose (FIG. 4). Aconcentration-dependent response was observed across all sucroseconcentrations evaluated. Notably there was a 24% increase in Man 5species, 12% increase in Man 6 species, and a 7% increase in Man 7species at the 70 mM sucrose supplementation condition. This cumulativeincrease of 43% in overall mannosylated N-glycans was accompanied by a51% drop in NGA2F species, the majority of which was precluded frombeing formed due to the abrogation of the N-glycan biosynthetic reactionas shown in FIG. 2. The data also demonstrate that combining the 51%decrease in NGA2F species and the 7% increase in NGA2F-GlcNAc speciesresults in an overall decrease in the fucosylation level of 44%. Thissignificant drop in fucosylation is important as it has been shown thatdecreased fucosylation of N-glycans has a significant and positiveeffect on the overall level of ADCC response. These data demonstratethat the supplementation of sucrose into cell culture media is capableof significantly re-distributing the overall protein glycosylationprofile of the produced protein, and for some commercial proteintherapeutics, this is a desired product quality attribute.

There was also a significant impact on the overall N-glycan distributionwhen the cell culture media was supplemented with tagatose (FIG. 6).Similar to the sucrose supplemented cell culture conditions, there was aconcentration-dependent response across all of the concentrationsevaluated. The results indicate that there was at most a 10% increase inMan 5 species, 6% increase in Man 6 species, 3% increase in Man 7species, and a 1% increase in Man 8 species at the 70 mM tagatosesupplementation condition. This cumulative 20% increase in overallmannosylated N-glycans was accompanied by a 30% decrease in NGA2Fspecies. In addition, there was an overall 23% decrease in overallfucosylation levels. Comparison of the results of sucrose and tagatosesupplementation demonstrate that the percent increase of N-glycans waslower in the tagatose supplemented cultures as compared to the sucrosesupplemented cultures. However, the individual N-glycan species whichwere modulated, were similar between the sucrose and tagatosesupplemented cultures. Both sugars utilize the same mechanism tomodulate the protein N-glycosylation profile as the pattern ofmodulation of the individual N-glycans species was consistent betweenboth sugars.

Laboratory-Scale Bioreactor Confirmation of the Targeted Modulation ofProtein Glycosylation Profiles

3 L scale-down model bioreactors were utilized to verify the impact ofselect sugars on the resulting protein glycosylation profiles. Theconcentration of the sucrose and tagatose was chosen to minimize anypotential adverse impact on cell growth and productivity, but stillfacilitate a measurable modulation of the glycosylation profile. Cellculture process performance indicators were monitored and measuredthroughout the respective cultures. Viable cell density, cell viability,lactate, pCO₂, osmolality, harvest titer, and harvest N-glycanoligosaccharide data were measured and reported.

FIGS. 7A-7F highlight the cell culture performance results observedthrough the use of sucrose supplementation in the basal and feed medias.In laboratory-scale bioreactors there was no impact on viable celldensity or cell viability upon supplementation with 50 mM sucrose whencompared to the unsupplemented control condition. Dissolved CO₂ (pCO₂)and lactate production are direct measures of the respiratory andmetabolic activities of mammalian cells, respectively. There was nosignificant difference in pCO₂ between the sucrose supplemented andnon-supplemented cultures, indicating no net change in the overallrespiratory activity of the cells. With respect to lactate, there wasonly a nominal increase in overall levels throughout the sucrosesupplemented culture, which is consistent with the overall higher levelof residual sugar in the culture media. Osmolality of the sucrosesupplemented condition was only slightly higher compared to the controlmostly due to the additional sugar solute. Upon harvest of the cultures,the final titer ratio of the sucrose supplemented culture was 0.78suggesting a slight drop in overall antibody productivity with inclusionof 50 mM sucrose. These data are consistent with the results observedfrom the shake flask cultures. After Protein A purification, theN-glycan glycoform profile was measured (FIG. 8). Similar to the shakeflask results, there was a significant increase in overall mannosylationlevels; Man 7 increased 3%, Man 6 increased 5%, and Man 5 increased 16%.This 24% increase in overall mannosylation was accompanied by a 29%decrease in NGA2F species, and an overall 24% decrease in fucosylation.These changes are very significant and are consistent with the resultsobserved in the shake flasks.

FIGS. 9A-9F highlight the cell culture results observed with the use oftagatose supplementation in the basal and feed medias. Tagatose had amuch more pronounced effect on viable cell density as compared to theunsupplemented control. The results indicated a significant reduction inpeak VCD from 11.2×10⁶ cells/mL for the control unsupplemented cultureversus 8.5×10⁶ cells/mL for the tagatose supplemented culture. Despitethis lower cell growth, cell viability remained high throughout theculture, and comparable to the results observed in the control culture.There was no significant difference in pCO₂ between the tagatosesupplemented and non-supplemented cultures, suggesting no net change inthe overall respiratory activity of the cells. With respect to lactate,there was only a nominal increase in overall levels throughout thetagatose supplemented culture, which is consistent with the overallhigher level of residual sugar in the culture media. Osmolality of thetagatose supplemented culture was higher as compared to the controlculture mostly due to the additional sugar solute. Upon harvest of thecultures, the final titer ratio of the tagatose supplemented culture was0.90 suggesting a slight drop in overall antibody productivity uponinclusion of 50 mM tagatose. The results were consistent with theresults observed from the shake flask cultures. The final harvest titerratio of the tagatose supplemented cultures was higher than the finalharvest titer ratio observed from the sucrose supplemented culture.

After Protein A purification, the N-glycan glycosylation profile wasmeasured with the results shown in FIG. 10. Similar to the shake flaskresults, there was a significant increase in overall mannosylationlevels; Man 7 increased 1%, Man 6 increased 2%, and Man 5 increased 7%.This 10% increase in overall mannosylation was accompanied by a 15%decrease in NGA2F species, and an overall 11% decrease in fucosylation.These changes are very significant and are consistent with the resultsobserved in the shake flasks.

The cell culture process performance results in 3 L-scale bioreactorsdemonstrated that the observed effect on protein glycosylation in shakeflasks is scale-independent. The significant increase in mannosylation,and resulting decrease in fucosylation has important implications forthe optimization of the production of protein therapeutics and themodulation of protein glycosylation profiles. Indeed, the selective useof sugars such as sucrose and tagatose increases the ability of cellculture scientists to customize the product characteristics ofrecombinant glycoproteins expressed in mammalian cells, and theresulting therapeutic activity, efficacy, and PK.

Evaluation of Alternative Cell Lines for the Impact of Selective SugarSupplementation on the Resulting Protein Glycosylation Profiles

Cell Line 2 was evaluated in shake flask culture in semi-fedbatch mode.Sucrose was supplemented into the basal media at various concentrationsto evaluate the resulting impact on cell culture performance, as well asthe protein glycosylation profile. The viable cell density, viability,and harvest titer results are shown in FIGS. 11A-11C. On average, 7 mM,15 mM, and 30 mM sucrose all nominally impacted overall cell growth,with only slight drops in peak viable cell density (1×10⁶-2×10⁶cells/mL) as compared to the unsupplemented control. Cell viability andharvest titer results were not significantly different from each other.These results are consistent with the lower concentration sucrosesupplementation results generated using Cell Line 1, in that at certainconcentrations, there is no significant impact on overall cell growth orproductivity. There was however, a very significant impact on theprotein glycosylation profiles of harvest samples across all sucroseconcentrations evaluated (FIG. 12). A concentration-dependent modulationof protein glycosylation was observed that was consistent with theresults obtained for Cell Line 1.

Notably there was up to a 17% increase in Man 5 species, 1% increase inMan 6 species, and a 1% increase in Man 7 species at the 30 mM sucrosesupplementation condition. This cumulative increase of 19% in overallmannosylated N-glycans was accompanied by a 27% drop in NGA2F species,and a 19% drop in overall fucosylation. Hence, as demonstrated with CellLine 1, there was a very significant impact on the N-glycan glycoformprofile. Specifically, individual N-glycans increased or decreased withsucrose supplementation of Cell Line 2 that was consistent with theresults obtained for Cell Line 1.

Example 2 1. Materials & Methods

Cell Culture

Two recombinant Chinese Hamster Ovary (CHO) cell lines expressing twodifferent recombinant glycoproteins were evaluated in two differentcultures vessels (shaker flasks and 3 L laboratory scale bioreactors).Cell Line 1 expressed Antibody 1, Cell Line 2 expressed Dual VariableDomain Immunoglobulin 1 (DVD 1) and Cell Line 3 expressed Antibody 2.Antibodies 1 and 2 are IgG1 proteins, and DVD 1 is an immunoglobulinwith two variable domains as documented previously (Wu C. et al., (2007)Nat. Biotechnol 25(11):1290-1297). All cell lines were of CHO DUX-B11origin based on a dhfr (dihydrofolate reductase) expression system. Allcell lines were cultured in the same chemically defined basal media.Cell lines 1 and 2 were fed with the same chemically-defined feed media,but Cell Line 1 was fed media that was formulated at a 50% higherconcentration. Each of the respective media were supplemented witheither sucrose or tagatose to evaluate their potential impact on theresulting N-glycan oligosaccharide profile. All sugars were purchasedfrom Sigma-Aldrich (St. Louis, Mo.). In preparation of the cultures, thecell lines were serially expanded through separate seed train inoculumsto generate enough cells for inoculation. Process conditions utilizedduring the cultures were slightly different between the two differentcell lines as shown in Table 1, but similar between each cell line andthe respective sugar supplemented control conditions.

TABLE 2 Summary of cell culture process conditions and sugarsupplementation details Cell Line 1 Cell Line 2 Cell Line 3 CultureVessel 250 mL 3 L lab-scale 250 mL 250 mL shaker flask bioreactorsshaker flask shaker flask Culture Mode Fedbatch Fedbatch FedbatchExtended Batch^(a) Initial Culture 36 36 35 36 Temperature (° C.)Dissolved N/A^(b) 30 N/A^(b) N/A^(b) Oxygen (%) pH N/A^(b) 6.9 N/A^(b)N/A^(b) Sugar Sucrose Sucrose Sucrose Sucrose Supplements TagatoseTagatose Tagatose Evaluated^(b) Fructose Supplement 0, 1, 10, 30, 0, 500, 1, 30, 50 0, 7, 15, 30 Concentrations 50, 70 (mM)^(c) ^(a)No feedmedia used; glucose added to cultures as needed to preclude glucosedepletion. ^(b)Cultures run in CO2 incubators at 5% CO2 in air; pH andDO parameters were not controlled, and thus do not have setpoint values.^(c)Supplements added to both chemically-defined basal and feed media.

Viable cell density (VCD) and cell viability values were measuredthrough trypan blue exclusion via Cedex automated cell counters (RocheApplied Science, Indianapolis, Ind.), glucose and lactate values weremeasured with a ABL-805 (Radiometer Medical, Denmark) blood gasanalyzer. Offline pH, dissolved oxygen (DO), and pCO₂ measurements wereperformed with an ABL-805 (Radiometer Medical, Denmark) blood gasanalyzer. Osmolality was measured on a Multi-Osmette 2430 osmometer(Precision Systems, Natick, Mass.).

Protein A Affinity Chromatography

Antibody titers were measured from crude cell culture harvests on aPoros A™ (Life Technologies, Carlsbad, Calif.) affinity column using anAgilent (Santa Clara, Calif.) 1200 Series HPLC, or equivalent, operatingwith a low pH, step elution gradient with detection at 280 nm. Absoluteconcentrations were assigned with respect to reference standardcalibration curves.

Purified antibodies subjected to additional analytical characterizationwere purified using MabSelect™ Protein A (GE Healthcare, Piscataway,N.J.) using a low pH, step elution gradient, followed by buffer exchangeusing Corning Lifesciences (Tewksbury, Mass.) Spin Concentrator X UFcolumns according to the manufacturers' recommended procedures.

N-Glycan Oligosaccharide Profiling

Approximately 200 μg of Protein A purified samples from Cell Lines 1 and2 were treated with N-glycanase at 37° C. overnight to remove theN-glycans from the protein. The protein was precipitated and thesupernatant was taken for subsequent chemical derivatization of thereducing end of the released glycans with 2-aminobenzamide (2-AB) dye.Following the derivatization step, the excess label was removed usingclean up cartridges and the samples were analyzed using normal phaseHPLC with fluorescent detection. Mobile phase A was 100% acetonitrileand mobile phase B was 50 mM ammonium formate pH 4.4. The glycans wereeluted from a polyamide column (Prozyme, Hayward, Calif.) using ashallow gradient. The labeled glycans were detected using a fluorescencedetector with an excitation wavelength of 330 nm and an emissionwavelength of 420 nm.

ADCC Measurements

ADCC activity was assessed using a ⁵¹Cr release assay. Approximately 1million cells expressing the target for Antibody 2 were labeled with 50μCi ⁵¹Cr for 1 hour at 37° C., washed with culture medium, and thenplated at 1×10⁴/well in a 96-well v-bottom plate. Antibody 2 wasincubated with target cells for 30 minutes at 4° C. before addition ofPBMC effector cells. After a 4 hour incubation at 37° C., 100 μL ofsupernatant were collected from each well, and released radioactivitywas counted with a Wallac WIZARD Gamma Counter (Perkin-Elmer). Allmeasurements were expressed as a percentage of total cell lysis.

Statistics

Experimental results are expressed as mean±SD for those resultsgenerated from at least three independent cultures. Experimental resultsare expressed as the measured value for those results generated fromless than three independent cultures. Results were evaluated forstatistical significance through 2-sided t-tests, with a requirement ofp<0.05 relative to the unsupplemented control conditions.

2. Results

Impact of Sucrose and Tagatose on the Protein Glycosylation Profiles ofRecombinant Antibodies

Cell Line 1 was cultured in shake flasks in fedbatch mode after anabbreviated seed train. Sucrose and tagatose were supplemented intochemically-defined basal and feed media at concentrations of 1, 10, 30,50, and 70 mM, and compared to unsupplemented control conditions. Theviable cell density (VCD), viability, and harvest titer results areshown in FIGS. 13A-13C for the sucrose supplemented cultures and FIGS.14A-14C for the tagatose supplemented cultures.

Amongst the sucrose supplemented cultures, viable cell density remainedessentially the same over time across the 1, 10, and 30 mM sucroseconditions. Only the 50 mM and 70 mM sucrose cultures demonstrated areduction in peak VCD, which were modest changes, but statisticallysignificant. Cell viability results were similar across the variousconditions up to process day 10. After process day 10, the 10, 30, 50,and 70 mM sucrose cultures all died slower than the control, and theresults were statistically significant. It is likely that the highersugar concentration in these cultures was responsible for the prolongedcell viability. Despite these changes in VCD and viability, the Antibody1 harvest titer results were essentially indistinguishable from thecontrol conditions. Only the 50 mM sucrose conditions demonstrated a 2%increase in harvest titer. Thus, across the range of testedconcentrations, sucrose is well tolerated by mammalian cells in culture,with only a minor impact on cell growth, and no impact on proteinproductivity.

The impact of sucrose on the N-glycan oligosaccharide distribution isshown in FIG. 13D. There was a significant impact on the overallN-glycan distribution. A concentration-dependent response was observedacross all the sucrose concentrations evaluated. Notably there was a 19%increase in Man 5 species, 10% increase in Man 6 species, 6% increase inMan 7 species, and 2% increase in Man 8 species for the 70 mM sucrosesupplementation condition. This cumulative increase of 37% in overallmannosylated N-glycans was accompanied by a 44% drop in NGA2F species,the majority of which was precluded from being formed due to theabrogation of the N-glycan biosynthetic reaction shown in FIG. 2. Thedata also demonstrate that the 44% decrease in NGA2F species, combinedwith a 1% decrease in NA1F species, and an 8% increase in NGA2F-GlcNAcspecies, resulted in an overall decrease in the fucosylation level of37%. The 10, 30, and 50 mM sucrose cultures demonstrated similarbehavior, but with lower absolute percent changes. However, the overallN-glycan core fucosylation was decreased in a statistically significantmanner at 10, 30, 50 and 70 mM sucrose, which is beneficial for somerecombinant proteins. The results also establish that sucrosesupplementation is a new powerful method for the re-distribution ofN-glycans towards high mannose glycans, which is a desirable productcharacteristic for some protein therapeutics.

Amongst the tagatose supplemented cultures, viable cell density remainedsimilar or higher over time across the 1 mM and 10 mM conditions.However, the 30, 50, and 70 mM tagatose cultures demonstrated areduction in peak VCD across many process days which was statisticallysignificant. Similar to the sucrose supplementation results, cellviability remained high throughout each of the tagatose supplementedcultures. The lower concentrations of 1 mM and 10 mM behaved in a mannersimilar to the control cultures. After process day 10, the 30, 50, and70 mM tagatose cultures all died slower than the control and the resultswere statistically significant across many of the days. It is likelythat the higher sugar concentration in these cultures was responsiblefor the higher cell viability. Despite these changes in VCD andviability, the Antibody 1 harvest titer results were essentiallyindistinguishable from the cells cultured under control conditions. Onlythe 70 mM tagatose conditions demonstrated a 9% decrease in harvesttiter. Thus, across the range of tested concentrations it can beconcluded that tagatose is well tolerated by mammalian cells in culture,with only a minor impact on cell growth and protein productivity.

The impact of tagatose on the N-glycan oligosaccharide distribution isshown in FIG. 14D. There was a significant impact on the overallN-glycan distribution. A concentration-dependent response was observedacross many of the tagatose concentrations evaluated. Notably there wasan 8% increase in Man 5 species, 4% increase in Man 6 species, 2%increase in Man 7 species, and 1% increase in Man 8 species for the 70mM tagatose supplementation condition. This cumulative increase of 15%in overall mannosylated N-glycans was accompanied by a 23% drop in NGA2Fspecies, the majority of which was precluded from being formed due tothe abrogation of the N-glycan biosynthetic reaction shown in FIG. 2.The data also demonstrate that the 23% decrease in NGA2F species,combined with a 8% increase in NGA2F-GlcNAc species, resulted in anoverall decrease in the fucosylation level of 15%. The 50 mM tagatosecultures demonstrated similar behavior, but with lower absolute percentchanges. The 1, 10, and 30 mM tagatose cultures provided for N-glycanprofiles that were not practically different compared to the controlculture. The results obtained with tagatose supplementation of cellculture media are similar to the results obtained with sucrosesupplementation. Supplementation of cell culture media with tagatoseresults in an overall decrease in core N-glycan fucosylation withpotential benefits towards ADCC, and provides a novel method for themodulation of N-glycan species, as well as a method for achievingproduct comparability with a reference protein.

Comparison of the results of sucrose or tagatose supplementationdemonstrates that the extent of change in the percent of N-glycans waslower in tagatose supplemented cultures compared to sucrose supplementedcultures. However, the specific N-glycan species which changed wassimilar between both conditions. Both sucrose and tagatose utilize thesame mechanism for effecting the protein N-glycosylation profile sincethe pattern of change of the individual N-glycans was consistent betweensupplementation with both sugars.

Impact of Sucrose and Tagatose on the Protein Glycosylation Profiles ofDual Variable Domain Immunoglobulins

Cell Line 2 was cultured in shake flasks in fed batch mode after anabbreviated seed train. Sucrose and tagatose were supplemented intochemically-defined basal and feed media at concentrations of 1, 30, and50 mM, and process performance results were compared to unsupplementedcontrol conditions. The viable cell density (VCD), viability, andharvest titer results are shown in FIGS. 15A-15C for the sucrosesupplemented cultures and FIGS. 16A-16C for the tagatose supplementedcultures.

Amongst the sucrose supplemented cultures, viable cell density remainedapproximately the same over time across the evaluated conditions. Onlythe 50 mM sucrose cultures demonstrated a reduction in peak VCD by atmost 2×10⁶ cells/mL. Cell viability results were similar across thevarious conditions up to process day 10. However, the relative harvesttiter results were different among the conditions tested. As the sucroseconcentration increased, the harvest titers decreased. The 50 mM sucrosecultures demonstrated the largest titer reduction of 48%, which wasstatistically significant from the control, and a much larger reductionthan observed in Cell Line 1. Thus, it appears that sucrose is not aswell tolerated in Cell Line 2 as compared to Cell Line 1.

The impact of sucrose on the N-glycan oligosaccharide distribution isshown in FIG. 15D. There was a significant impact on the overallN-glycan distribution with sucrose supplementation. Aconcentration-dependent response was observed across all sucroseconcentrations evaluated. Notably there was a 7% increase in Man 5species and a 1% increase in Man 6 species with 50 mM sucrosesupplementation of the culture. This cumulative increase of 8% inoverall mannosylated N-glycans was accompanied by a 20% drop in NGA2Fspecies, the majority of which was precluded from being formed due tothe abrogation of the N-glycan biosynthetic reaction shown in FIG. 2.The data also demonstrate that the 20% decrease in NGA2F species,combined with a 1% decrease in NA1F species, and a 8% increase inNGA2F-GlcNAc species, resulted in an overall decrease in thefucosylation level of 13%. The 30 mM sucrose cultures demonstrated asimilar decrease in overall fucosylation level, however the absolutepercent change was lower.

Tagatose was also evaluated with Cell Line 2 to evaluate the effect onprotein glycosylation. Similar to sucrose, none of the evaluatedconcentrations adversely impacted the viable cell density (VCD). At allconcentrations evaluated, the VCD was actually slightly higher comparedto the control. The cell viability results indicate that this parameterremained as high as the control through the majority of the process.After Day 8, the viability of the 30 mM and 50 mM tagatose supplementedcultures remained higher than the control, likely for the same reason asdiscussed above; the higher sugar concentration provided an enhancedlevel of nutrients that supported longer cell life. 1 mM tagatose didnot adversely impact relative harvest titer, but both 30 mM and 50 mMtagatose significantly decreased harvest titer by 15% and 38%,respectively.

The impact of tagatose on the N-glycan oligosaccharide distribution ofDVD 1 is shown in FIG. 16D. There was a significant impact on theoverall N-glycan distribution. A concentration-dependent response wasobserved across all the tagatose concentrations evaluated. Notably therewas a 3% increase in Man 5 species and a 1% increase in Man 6 speciesfor the 50 mM sucrose supplementation condition. This cumulativeincrease of 4% in overall mannosylated N-glycans was accompanied by a 9%decrease in NGA2F species, the majority of which was precluded frombeing formed due to the abrogation of the N-glycan biosynthetic reactionshown in FIG. 2. The data also demonstrate that the 9% decrease in NGA2Fspecies, combined with a 1% decrease in NA1F species, and a 5% increasein NGA2F-GlcNAc species, resulted in an overall decrease of thefucosylation level of 5%. The 30 mM tagatose cultures demonstrated asimilar decrease in overall fucosylation, however the absolute percentchange was lower.

Overall, sucrose and tagatose supplementation did not have as pronouncedof an impact on the N-glycan profile of DVD 1 as did the supplementationon Antibody 1. The differences in the structure of these proteinscontributed to the effect of sucrose or tagatose supplementation on theN-glycosylation profiles. These results demonstrate that while theabsolute percentage of various N-glycan species may vary, overall, theeffect of sucrose and/or tagatose supplementation on the N-glycanoligosaccharide profile is independent of the particular protein.

Impact of Sucrose and Tagatose on Protein Glycosylation Profiles isIndependent of Culture Scale

3 L scale laboratory bioreactors were utilized to verify the impact ofsucrose, tagatose, or fructose on protein glycosylation profiles.Concentrations of the sugars were chosen to minimize any potentialadverse impacts on cell growth and productivity, but still facilitate ameasurable impact on the glycoform profile. Cell culture processperformance indicators were monitored and measured throughout the study.Viable cell density, cell viability, lactate, pCO₂, osmolality, harvesttiter, and harvest N-glycan oligosaccharide data was measured andreported.

FIG. 17 shows the cell culture performance observed through the use ofsucrose, tagatose, or fructose supplementation into the basal and feedmedias. In laboratory-scale bioreactors there was no impact on viablecell density (VCD) or cell viability upon supplementation with 50 mMsucrose or 50 mM fructose compared to the unsupplemented controlculture. Tagatose had a much more pronounced effect on viable celldensity compared to the unsupplemented control. The results indicate asignificant reduction in peak VCD of 11.2×10⁶ cells/mL for the controlversus 8.5×10⁶ cells/mL for the tagatose supplemented culture. Despitethis lower cell growth, cell viability remained high throughout theculture, on par with the results observed with the control. Residualmedia glucose and lactate production are direct measures of themetabolic activities of mammalian cells, respectively. The three sugarsevaluated, all facilitated a higher relative level of residual glucoselevels compared to the unsupplemented control where glucose was the onlymedia sugar. With respect to lactate, there was no discernibledifference between the various cultures. Osmolality of the sucrose,tagatose, or fructose supplemented cultures were only slightly highercompared to the control, primarily due to the additional sugar solute.Upon harvest, the final titer ratio was 0.90 for the tagatosesupplemented culture, 0.78 for the sucrose supplemented culture, and0.81 for the fructose supplemented culture.

After Protein A purification, the N-glycan oligosaccharide profile wasmeasured with the results provided in FIG. 18. Similar to the shakeflask results, there was a significant increase in overall mannosylationlevels with sucrose supplementation. Man 7 increased 3%, Man 6 increased5%, and Man 5 increased 16% for the sucrose supplemented culture. This24% increase in overall mannosylation was accompanied by a 29% decreasein NGA2F species, 1% increase in NA1F-GlcNAc, 3% decrease in NA1F, 7%increase in NGA2F-GlcNAc, for an overall 24% decrease in fucosylation.The tagatose supplemented culture results were similar to those observedin the shake flasks. Man 7 increased 1%, Man 6 increased 2%, and Man 5increased 7%. This 10% increase in overall mannosylation was accompaniedby a 15% decrease in NGA2F species, 1% increase in NA1F-GlcNAc, 3%decrease in NA1F, and 6% increase in NGA2F-GlcNAc, for an overall 11%decrease in fucosylation. These changes in N-glycan oligosaccharideprofile were consistent with the results obtained using the shakeflasks.

The fructose supplemented culture did not facilitate any significantchanges in protein glycosylation compared to the control. It isinteresting that the results observed with sucrose supplementation, whenglucose is already in the media, do not agree with the results observedwith fructose supplementation into the cell culture media. Sucrose,which is comprised of glucose and fructose, modulates a proteinglycosylation profile that does not correspond to the profiledemonstrated when glucose and/or fructose are the predominant sugars inthe cell culture. This indicates that the mechanism for sucrosemodulation of the protein glycosylation profile (e.g., the increase inhigh mannose N-glycans and decrease in the fucosylation level) is notdue to its constituent fructose or glucose alone. Instead, it is thecomplex of glucose to fructose (i.e., sucrose) that facilitates anenzymatic block at the high mannose to complex type N-glycanbiosynthetic pathway (shaded areas in FIG. 2).

The cell culture process performance results in 3 L-scale bioreactorsdemonstrate that the effect of sucrose or tagatose supplementation onprotein glycosylation is volumetric scale-independent. The significantincrease in mannosylation, and resulting decrease in fucosylation, hasimportant implications for the optimization of the production of proteintherapeutics and the modulation of protein glycosylation profiles.Indeed, the selective use of sugars, such as sucrose and tagatose,increases the ability to customize the product characteristics ofrecombinant glycoproteins expressed in mammalian cells, and theresulting therapeutic activity, or comparability to a reference protein.

High Mannosylation/Low Fucosylation Effect on ADCC

Cell Line 3 was evaluated in shake flask culture in chemically definedmedia supplemented with 7, 15, and 30 mM sucrose. The cultures wereharvested, and the N-glycan oligosaccharide profiles were measured withthe results shown in FIG. 20. There was a significant increase inoverall mannosylation levels, and thus a significant decrease in overallfucosylation levels. The response to sucrose was dependent on thesucrose concentration and similar to the response demonstrated in CellLines 1 and 2. Notably there was a 17% increase in Man 5 species, 1%increase in Man 6 species, and a 1% increase in Man 7 species for the 30mM sucrose supplementation condition. This cumulative increase of 19% inoverall mannosylated N-glycans was accompanied by a 27% drop in NGA2Fspecies, the majority of which was precluded from being formed due tothe abrogation of the N-glycan biosynthetic reaction shown in FIG. 2.The data also demonstrates that with the combined 27% decrease in NGA2Fspecies and 8% increase in NGA2F-GlcNAc species, that the overallfucosylation level was decreased by 19%. Antibody 3, purified from thesucrose supplemented cell cultures, was tested in a Cr-51 release assayto evaluate effector function, specifically ADCC activity. This wasperformed to verify that the lower level of fucosylated N-glycans linkedto Antibody 3 would facilitate an increase in ADCC activity. The resultsare shown in FIG. 21. Antibodies purified from the 30 mM sucrosesupplemented culture had the highest levels of high mannose N-glycans,and lowest levels of fucosylated N-glycans. These antibodies alsodemonstrated the largest percent lysis of target cells, an increase ofabout 15% compared to the cell lysis demonstrated with theunsupplemented control. Purified antibodies from the culturessupplemented with lower sucrose concentrations of 7 mM and 15 mM alsofacilitated an increase in percent lysis of target cells relative to thecontrol, though the increase was lower when compared to the increase inpercent lysis produced by antibodies purified from the culturessupplemented with 30 mM sucrose. These results indicate that not onlycan sucrose supplementation modulate the glycosylation profile ofproteins, e.g., antibodies, but the modulation has a beneficial impacton ensuring biologic comparability as well as therapeutic efficacy.

Discussion

Sucrose and tagatose are two sugars found in nature. Sucrose, inparticular, has been found to be the most abundant sugar in soy beans(Hou A., et al., Internat. J Agronomy (2009) Article ID 484571:8).

The data provided above demonstrate that the selective supplementationof sucrose and tagatose in host cell culture media is an effectiveapproach for the targeted modulation of protein glycosylation profilesin cultured cell lines. In particular, supplementation of host cellculture media effectively modulated the N-glycan glycoform profiletowards high mannose species, and decreased the overall level offucosylated species. Since both sugars resulted in similar glycosylationprofiles, it is likely that they modulate glycosylation via the samemechanism or pathway. Of the high mannose N-glycan species, Man 5 glycanincreased the most upon exposure to either of the two sugars. Amongstthe non-mannosylated N-glycan species, the largest increase wasdemonstrated in NGA2F-GlcNAc levels. The fact that both of Man 5 glycanand NGA2F-GlcNAc share the same UDP-GlcNAc co-substrate (FIG. 2,highlighted) suggests that their accumulation may be due to inhibitionof these reactions through a diminished supply of available UDP-GlcNAcsubstrate. The same shifting in protein glycosylation profiles wasobserved across multiple sucrose and tagatose concentrations in aconcentration-dependent manner and across multiple cell lines expressingrecombinant proteins. At select concentrations, an adverse impact ofthese sugars on cell growth and productivity was minimized. The use ofsucrose and/or tagatose in cell culture media provides an efficient andeffective approach toward re-targeting of specific N-glycan glycoformprofiles. This capability is important for ensuring biologiccomparability, as well as the targeted optimization of product quality.The targeted increase in mannosylation levels, and decrease infucosylation levels, is an important capability since for somerecombinant glycoprotein therapeutics, high mannose glycans are adesired product characteristic. Furthermore, the decrease in overallfucosylation levels has been correlated with an increased ADCC response.High mannose glycans are also reported to be cleared faster fromcirculation (Alessandri, L., et al., (2012) mAbs Journal 4(4): 1-1210).The ability to modulate protein glycosylation through the selective useof monosaccharides and oligosaccharides (e.g., sucrose and tagatose) iscritically important for achieving critical product characteristics.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and theaccompanying figures. Such modifications are intended to fall within thescope of the appended claims.

Patents, patent applications, publications, product descriptions,GenBank Accession Numbers, and protocols that may be cited throughoutthis application, the disclosures of which are incorporated herein byreference in their entireties for all purposes. The contents of allcited references, including literature references, issued patents, andpublished patent applications, as cited throughout this application arehereby expressly incorporated herein by reference. It should further beunderstood that the contents of all the figures and tables attachedhereto are expressly incorporated herein by reference. The entirecontents of the following applications are also expressly incorporatedherein by reference: U.S. Provisional Patent Application 61/893,123,entitled “STABLE SOLID PROTEIN COMPOSITIONS AND METHODS OF MAKING SAME”,Attorney Docket Number 117813-31001, filed on Oct. 18, 2013; U.S.Provisional Application Ser. No. 61/892,833, entitled “LOW ACIDICSPECIES COMPOSITIONS AND METHODS FOR PRODUCING THE SAME USINGDISPLACEMENT CHROMATOGRAPHY”, Attorney Docket Number 117813-73602, filedon Oct. 18, 2013; U.S. Provisional Patent Application 61/892,710,entitled “MUTATED ANTI-TNFa ANTIBODIES AND METHODS OF THEIR USE”,Attorney Docket Number 117813-73802, filed on Oct. 18, 2013; U.S.Provisional Patent Application 61/893,088, entitled “MODULATED LYSINEVARIANT SPECIES AND METHODS FOR PRODUCING AND USING THE SAME”, AttorneyDocket Number 117813-74101, filed on Oct. 18, 2013; U.S. ProvisionalPatent Application 61/893,131, entitled “PURIFICATION OF PROTEINS USINGHYDROPHOBIC INTERACTION CHROMATOGRAPHY”, Attorney Docket Number117813-74301, filed on Oct. 18, 2013; and U.S. patent application Ser.No. 14/077,871, entitled “LOW ACIDIC SPECIES COMPOSITIONS AND METHODSFOR PRODUCING AND USING THE SAME”, Attorney Docket Number 117813-73902,filed on Nov. 12, 2013.

We claim:
 1. A method of producing a composition comprising a proteinwith a modulated glycosylation profile, said method comprising:culturing a host cell expressing said protein in cell culture mediasupplemented with a monosaccharide and/or an oligosaccharide, therebyproducing said composition comprising said protein with a modulatedglycosylation profile as compared to a control, wherein said control isa composition comprising a protein produced by culturing a host cellexpressing said protein in cell culture media which is not supplementedwith a monosaccharide and/or an oligosaccharide.
 2. The method of claim1, further comprising purifying said composition comprising said proteinwith a modulated glycosylation profile.
 3. The method of claim 1,wherein the protein is an antibody or antigen-binding portion thereof.4. The method of claim 3, wherein the antibody is an anti-TNFα antibody.5. The method of claim 4, wherein the anti-TNFα antibody is adalimumab,or antigen binding fragment thereof.
 6. The method of claim 1, whereinthe protein is a dual variable domain immunoglobulin (DVD-Ig).
 7. Themethod of claim 1, wherein the protein is selected from the groupconsisting of a TVD-IG, a half-body and a RAB.
 8. The method of claim 1,wherein the monosaccharide is tagatose.
 9. The method of claim 1,wherein the oligosaccharide is sucrose.
 10. The method of claim 1,wherein the cell culture media is supplemented with a sufficient amountof the monosaccharide to achieve a monosaccharide concentration selectedfrom the group consisting of about 1 mM, about 5 mM, about 7 mM, about10 mM, about 20 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM,about 70 mM, about 80 mM, about 90 mM and about 100 mM.
 11. The methodof claim 10, wherein the monosaccharide concentration is 30 mM.
 12. Themethod of claim 10 or 11, wherein the monosaccharide is tagatose. 13.The method of claim 1 wherein the cell culture media is supplementedwith a sufficient amount of the oligosaccharide to achieve anoligosaccharide concentration selected from the group consisting ofabout 1 mM, about 5 mM, about 7 mM, about 10 mM, about 20 mM, about 30mM. about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM,about 90 mM and about 100 mM.
 14. The method of claim 13, wherein theoligosaccharide concentration is 30 mM.
 15. The method of claim 13 or14, wherein the oligosaccharide is sucrose.
 16. The method of claim 1,wherein the modulated glycosylation profile of the protein comprisesmodulation of a fucosylation level and/or a mannosylated N-glycanoligosaccharide level in said protein.
 17. The method of claim 16,wherein the modulation of the fucosylation level comprises a decrease inthe fucosylation level in said protein.
 18. The method of claim 17,wherein the decrease in the fucosylation level comprises a decrease inthe level of NGA2F, NA1F-GlcNAc, NA1F and/or NA2F in said protein. 19.The method of claim 18, wherein the decrease in the level of NGA2F,NA1F-GlcNAc, NA1F and/or NA2F is a decrease of about 0.1%, 1%, 1.2%,1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or 65%.
 20. The method of claim16, wherein the modulation of the fucosylation level comprises anincrease in the fucosylation level in said protein.
 21. The method ofclaim 20, wherein increase in the fucosylation level comprises anincrease in the level of NGA2F-GlcNAc, NA1F-GlcNAc, NA1F and/or NA2F insaid protein.
 22. The method of claim 20, wherein the increase in thelevel of NGA2F-GlcNAc, NA1F-GlcNAc, NA1F or NA2F is an increase of about0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%,5%, 10%, 15% or 20%.
 23. The method of claim 17, wherein the overalldecrease in the fucosylation level comprises an increase or a decreasein the level of NGA2F, NGA2F-GlcNAc, NA1F-GlcNAc, NA1F and/or NA2F insaid protein.
 24. The method of claim 23, wherein in the overalldecrease in the fucosylation level is a decrease of about 1%, 1.2%,1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45% or 50%.
 25. The method of claim 16, wherein themodulation of the mannosylated N-glycan level comprises an increase inthe mannosylation level of said protein.
 26. The method of claim 25,wherein the increase in the mannosylation level is an increase of about0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%, 4%, 4.2%, 4.5%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.
 27. The method ofclaim 25, wherein the increase in the mannosylation level comprises anincrease in the level of a high mannose N-glycan oligosaccharideselected from the group consisting of Man 5 glycan, Man 6 glycan, Man 7glycan and Man 8 glycan.
 28. The method of claim 27, wherein the levelsof Man 5 glycan, Man 6 glycan, Man 7 glycan and/or Man 8 glycan areincreased by about 0.1%, 1%, 1.2%, 1.5%, 2%, 2.2%, 2.5%, 3%, 3.2%, 3.5%,4%, 4.2%, 4.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40, 45% or 50%. 29.The method of claim 1, wherein said host cell is a CHO cell.
 30. Amethod of producing a composition comprising an antibody, or antigenbinding fragment thereof, with a modulated glycosylation profile, saidmethod comprising: culturing a host cell expressing said antibody, orantigen binding fragment thereof, in cell culture media supplementedwith sucrose and/or tagatose, thereby producing said compositioncomprising said antibody, or antigen binding fragment thereof, with anincreased level of mannosylated N-glycans and a decreased level offucosylated N-glycans as compared to a control, wherein said control isa composition comprising an antibody, or antigen binding fragmentthereof, produced by culturing a host cell expressing said antibody, orantigen binding fragment thereof, in cell culture media which is notsupplemented with tagatose and/or glucose.
 31. The method of claim 30,wherein the antibody is adalimumab, or antigen binding fragment thereof.32. A method of producing a composition comprising an antibody, orantigen binding fragment thereof, with a modulated glycosylationprofile, said method comprising: culturing a host cell expressing saidantibody, or antigen binding fragment thereof, in cell culture mediasupplemented with sucrose and/or tagatose, thereby producing saidcomposition comprising said antibody, or antigen binding fragmentthereof, with a 1-50% increase in the level of mannosylated N-glycansand a 1-50% decrease in the level of fucosylated N-glycans as comparedto a control, wherein said control is a composition comprising anantibody, or antigen binding fragment thereof, produced by culturing ahost cell expressing said antibody, or antigen binding fragment thereof,in cell culture media which is not supplemented with tagatose and/orglucose.
 33. The method of claim 32, wherein the antibody is adalimumab,or antigen binding fragment thereof.
 34. A composition comprising a cellculture media comprising a monosaccharide and/or an oligosaccharide. 35.The composition of claim 34, wherein the monosaccharide is tagatose. 36.The composition of claim 34, wherein the oligosaccharide is sucrose. 37.A pharmaceutical composition comprising the composition produced by themethods of any one of claims 1, 30 or 32 and a pharmaceuticallyacceptable carrier.
 38. A composition comprising a therapeutic proteinwith a modulated glycosylation profile produced by the methods of anyone of claims 1, 30 or
 32. 39. The composition of claim 38, wherein thetherapeutic protein is an antibody.
 40. A composition comprising atherapeutic protein, wherein said protein comprises a 1-50% increase inthe level of mannosylated N-glycans and a 1-50% decrease in the level offucosylated N-glycans, as compared to a control, wherein said control isa composition comprising a protein produced by culturing a host cellexpressing said protein in cell culture media which is not supplementedwith a monosaccharide and/or an oligosaccharide
 41. The composition ofclaim 40, wherein the therapeutic protein is selected from the groupconsisting of an antibody, an antigen-binding portion thereof, DVD-Ig,TVD-Ig, RAB and half-body.
 42. A composition of claim 40, wherein thetherapeutic protein is an antibody.
 43. A method of producing acomposition comprising an antibody, or antigen binding fragment thereof,with a modulated glycosylation profile, said method comprising:culturing a host cell expressing said antibody, or antigen bindingfragment thereof, in cell culture media supplemented with sucrose and/ortagatose, thereby producing said composition comprising said antibody,or antigen binding fragment thereof, with a 1-30% increase inantibody-dependent cellular cytotoxicity (ADCC) response as compared toa control, wherein said control is a composition comprising an antibody,or antigen binding fragment thereof, produced by culturing a host cellexpressing said antibody, or antigen binding fragment thereof, in cellculture media which is not supplemented with tagatose and/or glucose.44. The method of claim 43, wherein the antibody is adalimumab, orantigen binding fragment thereof.